ResearchPad - paper https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[An outlook on livestock welfare conditions in African communities — A review]]> https://www.researchpad.co/article/elastic_article_14454 A significant proportion of the African continent is conducive for animal agricultural production, due to its historical experience and available resources to accommodate and nurture various indigenous and exotic animal species and breeds. With food security being a global challenge, animal products can play an important role as nutrient dense food sources in human diets, particularly in Africa. However, this does not seem to reach its full potential in practice, due to numerous reasons that have not been adequately addressed. Animal welfare reservations can be highlighted as one of the major contributing factors to the curbed progress. The consequences have been scientifically proven to affect product quality and market access. However, in the African community, the concept of animal welfare has not been fully embraced. While there are international animal welfare standards in the developed world, there are inherent factors that hinder adoption of such initiatives in most developing regions, particularly among communal farmers. These include cultural norms and practices, social ranking, socio-economic status, available resources, information dissemination and monitoring tools. Therefore, there is need to harmonize what is internationally required and what is feasible to accommodate global variability. The protocols followed to ensure and evaluate farm animal welfare require regular investigation, innovation and a sustainable approach to enhance animal productivity, efficiency and product quality. Additionally, investing in animal wellbeing and health, as well as empowering communities with significant knowledge, has a potential to improve African livelihoods and contribute to food security. This review seeks to highlight the concept of animal welfare in relation to livestock and food production in African conditions.

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<![CDATA[Effect of home-based Tai Chi, Yoga or conventional balance exercise on functional balance and mobility among persons with idiopathic Parkinson’s disease: An experimental study]]> https://www.researchpad.co/article/elastic_article_14425 Individuals with Parkinson’s disease (PD) invariably experience functional decline in a number of motor and non-motor domains affecting posture, balance and gait. Numerous clinical studies have examined effects of various types of exercise on motor and non-motor problems. But still much gap remains in our understanding of various therapies and their effect on delaying or slowing the dopamine neuron degeneration. Recently, Tai Chi and Yoga both have gained popularity as complementary therapies, since both have components for mind and body control.Objective:The aim of this study was to determine whether eight weeks of home-based Tai Chi or Yoga was more effective than regular balance exercises on functional balance and mobility.Methods:Twenty-seven individuals with Idiopathic PD (Modified Hoehn and Yahr stages 2.5–3) were randomly assigned to either Tai Chi, Yoga or Conventional exercise group. All the participants were evaluated for Functional Balance and Mobility using Berg Balance Scale, Timed 10 m Walk test and Timed Up and Go test before and after eight weeks of training.Results:The results were analyzed using two-way mixed ANOVA which showed that there was a significant main effect for time as F (1, 24) =74.18, p=0.000, ηp2=0.76 for overall balance in Berg Balance Scale. There was also significant main effect of time on mobility overall as F(1, 24) =77.78, p=0.000, ηp2=0.76 in Timed up and Go test and F(1, 24) =48.24, p=0.000, ηp2=0.67 for 10 m Walk test. There was a significant interaction effect for time×group with F(2, 24) =8.67, p=0.001, ηp2=0.420 for balance. With respect to mobility, the values F(2, 24) =5.92, p=0.008, ηp2=0.330 in Timed Up and Go test and F(2, 24) =10.40, p=0.001, ηp2=0.464 in 10 m Walk test showed a significant interaction. But there was no significant main effect between the groups for both balance and mobility.Conclusion:The findings of this study suggest that Tai Chi as well as Yoga are well adhered and are attractive options for a home-based setting. As any form of physical activity is considered beneficial for individuals with PD either Tai Chi, Yoga or conventional balance exercises could be used as therapeutic intervention to optimize balance and mobility. Further studies are necessary to understand the mind–body benefits of Tai Chi and Yoga either as multicomponent physical activities or as individual therapies in various stages of PD. ]]> <![CDATA[Experiences of occupational health doctors and nurses about the role of physiotherapists in occupational health rehabilitation: A qualitative study]]> https://www.researchpad.co/article/elastic_article_14424 Occupational health physiotherapy has been practiced in the UK over several decades. In the past decade, the role of occupational health physiotherapy has gained recognition as a profession that can be embedded within occupational health departments; however, limited information is known about the role of physiotherapists from professional groups outside the allied health domain in this context.Objective:The aim of this study is to explore the experiences of occupational health doctors and nurses about the role of physiotherapy in occupational health rehabilitation.Methods:This study is a qualitative investigation underpinned by an interpretative construct. Thirteen semi-structured interviews were conducted. Two occupational health doctors and 12 nurses were purposively recruited from two National Health Service (NHS) hospitals. Data were analyzed using thematic content analysis, coded manually and verified by member checking.Results:The benefits of occupational health physiotherapists were rapid access intervention, advanced knowledge and clinical reasoning, evidence-based practice, and providing an additional perspective. The emerging themes of the challenges that occupational health physiotherapists may face include dealing with occupational health challenges, managing role conflicts, personal qualities and attributes, and role substitution.Conclusion:Participants described numerous roles of occupational health physiotherapists ranging from clinical to organizational components. On-going research is needed to support the role development of physiotherapists providing occupational health rehabilitation and to further advocate for its relevance in this setting. ]]> <![CDATA[Responsiveness of pain, functional capacity tests, and disability level in individuals with chronic nonspecific low back pain]]> https://www.researchpad.co/article/elastic_article_14423 Clinical outcomes are very important in clinical assessment, and responsiveness is a component inside the outcome measures that needs to be investigated, particularly in chronic nonspecific low back pain (CNSLBP).Objective:This study aimed to investigate the responsiveness of pain, functional capacity tests, and disability in individuals with CNSLBP.Methods:Twenty subjects were assessed in pain using the following methods: visual analog scale (VAS) and numeric pain rating scale (NPRS), functional capacity tests: functional reach test (FRT), five-time sit-to-stand test (5 TSST), and two-minute step test (2 MST), and disability level: modified Oswestry Disability Questionnaire (MODQ), Thai version before and after 2-week intervention session. For interventions, the subjects received education, spinal manipulative therapy, and individual therapeutic exercise twice a week, for a total of two weeks. The statistics analyzed were change scores, effect size (ES), and standardized response mean (SRM).Results:The most responsive parameter for individuals with CNSLBP was pain as measured by numeric pain rating scale (NPRS) (ES −0.986, SRM −0.928) and five-time sit-to-stand test (5 TSST) (SRM −0.846).Conclusion:This study found that NPRS pain and 5 TSST were responsive in individuals with CNSLBP at two weeks after the beginning of interventions. ]]> <![CDATA[Intertester reliability of a movement impairment-based classification system for individuals with shoulder pain]]> https://www.researchpad.co/article/elastic_article_14422 Other than pathoanatomical diagnosis, physical therapy managements need the diagnosis of movement-related impairments for guiding treatment interventions. The classification system of the Movement System Impairment (MSI) has been adopted to label the musculoskeletal disorders in physical therapy practice. However, reliability study of this classification system in individuals with shoulder pain has not been reported in the literature.Objective:This paper investigated the intertester reliability of the diagnosis based on the MSI classification system in individuals with shoulder pain.Methods:The patients with shoulder pain, between the ages 18–60 years, were recruited if he or she had pain between 30 and 70 on the 100 mm visual analog scale for at least three months. The examiners who were two physical therapists with different clinical experiences received a standardized training program. They independently examined 45 patients in random order. Each patient was examined by both therapists on the same day. The standardized examination scheme based on the MSI approach was used. Patients were identified to subgroup syndromes according to scapular and humeral syndromes and also determining their subcategory syndromes. Six scapular subcategory syndromes included downward rotated, depressed, abducted, wing, internal rotated/anterior tilted, and elevated. Three humeral subcategory syndromes were anterior glide, superior glide, and medial rotated. More than one subgroup and subcategory of syndromes could be identified in each patient. The test results of each session were blinded to another therapist. The percentages of agreement and kappa statistic were determined.Results:The results showed that agreement levels in identifying subgroup syndromes was fair (71.11% agreement, kappa coefficient = 0.34) and classifying subcategories syndromes were poor to substantial (73.33–91.11% agreement, kappa coefficient = 0.20–0.66). The overall agreement and kappa value of the MSI classification of subcategory syndromes was poor (kappa coefficient = 0.11; 95% CI 0.05–0.18). The agreement level of subcategories for scapular depression and humeral superior glide syndromes was substantial. The scapular winging, depression, and downward rotation were the three syndromes that were most frequently identified by both the examiners.Conclusion:The intertester reliability between therapists with different experience according to the MSI approach for shoulder pain classification was generally acceptable to poor due to the nature of the classification system. The standardized procedure and intensive training can be used for inculcating novice therapists with adequate level of intertester reliability of examination. ]]> <![CDATA[Thai dance exercises benefited functional mobility and fall rates among community-dwelling older individuals]]> https://www.researchpad.co/article/elastic_article_14421 With dramatic increase in the number of older individuals, special efforts have been made to promote the levels of independence and reduce fall rates among these individuals.Objective:To investigate the effects of Thai dance exercises over 6 weeks on functional mobility and fall rates in community-dwelling older individuals.Methods:Sixty-one community-dwelling older adults were interviewed and assessed for their demographics and fall data during 6 months prior to participation in the study. Then they completed the quasi-experimental Thai dance exercise program for 50 minutes/day, 3 days/week over 6 weeks. Their functional mobility relating to levels of independence and safety were assessed prior to training, at 3-week and 6-week training. After completing the program at 6 weeks, participants were prospectively monitored for fall data over 6 months.Results:Participants improved their functional mobility significantly after 3- and 6-week training (p<0.01). The number of faller individuals obviously decreased from 35% (n=21) prior to training to only 8% (n=5) after training (p<0.01).Conclusion:The current findings further extend benefits of Thai dance as an alternative musical exercise program to promote levels of independence and safety among community-dwelling older adults. ]]> <![CDATA[Association between lumbopelvic motion and muscle activation in patients with non-specific low back pain during forward bending task: A cross-sectional study]]> https://www.researchpad.co/article/elastic_article_14420 Evidence suggests patients with non-specific low back pain (NSLBP) have altered lumbar and pelvic movement patterns. These changes could be associated with altered patterns of muscle activation.Objective:The study aimed to determine: (1) differences in the relative contributions and velocity of lumbar and pelvic movements between people with and without NSLBP, (2) the differences in lumbopelvic muscle activation patterns between people with and without NSLBP, and (3) the association between lumbar and pelvic movements and lumbopelvic muscle activation patterns.Methods:Subjects (8 healthy individuals and 8 patients with NSLBP) performed 2 sets of 3 repetitions of active forward bending, while motion and muscle activity data were collected simultaneously. Data derived were lumbar and pelvic ranges of motion and velocity, and ipsilateral and contralateral lumbopelvic muscle activities (internal oblique/transverse abdominis (IO/TA), lumbar multifidus (LM), erector spinae (ES) and gluteus maximus (GM) muscles).Results:Lumbar and pelvic motions showed trends, but exceeded 95% confidence minimal detectable difference (MDD95), for greater pelvic motion (p=0.06), less lumbar motion (p=0.23) among patients with NSLBP. Significantly less activity was observed in the GM muscles bilaterally (p<0.05) in the NSLBP group. A significant association (r=−0.8, p=0.02) was found between ipsilateral ES muscle activity and lumbar motion, while moderate, but statistically non-significant associations, were found between GM muscle activity bilaterally and lumbar velocity (ipsilateral: r=−0.6, p=0.14; contralateral: r=−0.6, p=0.16) in the NSLBP group.Conclusion:Findings indicated patients had greater pelvic contribution, but less lumbar contribution which was associated with less activation of the GM bilaterally. ]]> <![CDATA[Patients with <i>in-situ</i> metallic coils and Amplatzer vascular plugs used to treat pulmonary arteriovenous malformations since 1984 can safely undergo magnetic resonance imaging]]> https://www.researchpad.co/article/elastic_article_14394 To examine the MRI safety of metallic coils and Amplatzer vascular plugs. Currently, concern regarding MR safety of devices used to treat pulmonary arteriovenous malformations (PAVMs) causes delays in performing emergency MRI in patients presenting with acute neurological symptoms.Methods:A retrospective audit was performed on all patients who underwent PAVM embolization at Hammersmith Hospital, London UK between 1984 and 2017. Outcomes of all MRI studies performed at our institution were recorded. In addition, known outcomes of all known MRI studies performed on patients treated with the earliest steel coils (1984–1995) were recorded.Results:At our institution, 20 patients underwent 1.5 T MRI after the insertion of 100 steel coils (15.5 – 28.6, median 22 years later), 140 coils designated MR-conditional (0.42 – 12.7, median 9.3 years later), and 54 MRI-conditional Amplatzer vascular plugs (0.17 – 8.0, median 0.75 years later), many in combination. The majority of scans were for cerebral indications, but other body regions scanned included spinal, thoracic, and pelvic regions. No adverse events were reported. Similarly, there were no adverse events in any MR scan known to have been performed in other institutions in seven further patients treated with the earliest steel coils (1984–1995). Again, the majority of scans were for cerebral indications.Conclusion:The findings demonstrate MR safety at 1.5 T of all PAVM embolization devices inserted in a main UK centre since inception in 1984.Advances in knowledge:MRI of patients who have had PAVMs treated by embolization can be implemented without contacting specialist pulmonary arteriovenous malformation treatment centres for approval. ]]> <![CDATA[MR guided high intensity focused ultrasound (MRgHIFU) for treating recurrent gynaecological tumours: a pilot feasibility study]]> https://www.researchpad.co/article/elastic_article_14393 To assess the feasibility of targeting recurrent gynaecological tumours with MR guided high intensity focused ultrasound (MRgHIFU).Methods:20 patients with recurrent gynaecological tumours were prospectively scanned on a Philips/Profound 3 T Achieva MR/ Sonalleve HIFU system. Gross tumour volume (GTV) and planning target volume (PTV) were delineated on T 2W and diffusion-weighted imaging (DWI). Achievable treatment volumes that (i) assumed bowel and/or urogenital tract preparation could be used to reduce risk of damage to organs-at-risk (TVoptimal), or (ii) assumed no preparations were possible (TVno-prep) were compared with PTV on virtual treatment plans. Patients were considered treatable if TVoptimal ≥ 50 % PTV.Results:11/20 patients (55%) were treatable if preparation strategies were used: nine had central pelvic recurrences, two had tumours in metastatic locations. Treatable volume ranged from 3.4 to 90.3 ml, representing 70 ± 17 % of PTVs. Without preparation, 6/20 (30%) patients were treatable (four central recurrences, two metastatic lesions). Limiting factors were disease beyond reach of the HIFU transducer, and bone obstructing tumour access. DWI assisted tumour outlining, but differences from T 2W imaging in GTV size (16.9 ± 23.0%) and PTV location (3.8 ± 2.8 mm in phase-encode direction) limited its use for treatment planning.Conclusions:Despite variation in size and location within the pelvis, ≥ 50 % of tumour volumes were considered targetable in 55 % patients while avoiding adjacent critical structures. A prospective treatment study will assess safety and symptom relief in a second patient cohort.Advances in knowledge:Target size, location and access make MRgHIFU a viable treatment modality for treating symptomatic recurrent gynaecological tumours within the pelvis. ]]> <![CDATA[Risk factors for adverse clinical outcomes with COVID-19 in China: a multicenter, retrospective, observational study]]> https://www.researchpad.co/article/elastic_article_14338 Background: The risk factors for adverse events of Coronavirus Disease-19 (COVID-19) have not been well described. We aimed to explore the predictive value of clinical, laboratory and CT imaging characteristics on admission for short-term outcomes of COVID-19 patients.

Methods: This multicenter, retrospective, observation study enrolled 703 laboratory-confirmed COVID-19 patients admitted to 16 tertiary hospitals from 8 provinces in China between January 10, 2020 and March 13, 2020. Demographic, clinical, laboratory data, CT imaging findings on admission and clinical outcomes were collected and compared. The primary endpoint was in-hospital death, the secondary endpoints were composite clinical adverse outcomes including in-hospital death, admission to intensive care unit (ICU) and requiring invasive mechanical ventilation support (IMV). Multivariable Cox regression, Kaplan-Meier plots and log-rank test were used to explore risk factors related to in-hospital death and in-hospital adverse outcomes.

Results: Of 703 patients, 55 (8%) developed adverse outcomes (including 33 deceased), 648 (92%) discharged without any adverse outcome. Multivariable regression analysis showed risk factors associated with in-hospital death included ≥ 2 comorbidities (hazard ratio [HR], 6.734; 95% CI; 3.239-14.003, p < 0.001), leukocytosis (HR, 9.639; 95% CI, 4.572-20.321, p < 0.001), lymphopenia (HR, 4.579; 95% CI, 1.334-15.715, p = 0.016) and CT severity score > 14 (HR, 2.915; 95% CI, 1.376-6.177, p = 0.005) on admission, while older age (HR, 2.231; 95% CI, 1.124-4.427, p = 0.022), ≥ 2 comorbidities (HR, 4.778; 95% CI; 2.451-9.315, p < 0.001), leukocytosis (HR, 6.349; 95% CI; 3.330-12.108, p < 0.001), lymphopenia (HR, 3.014; 95% CI; 1.356-6.697, p = 0.007) and CT severity score > 14 (HR, 1.946; 95% CI; 1.095-3.459, p = 0.023) were associated with increased odds of composite adverse outcomes.

Conclusion: The risk factors of older age, multiple comorbidities, leukocytosis, lymphopenia and higher CT severity score could help clinicians identify patients with potential adverse events.

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<![CDATA[Super-resolution observation of lysosomal dynamics with fluorescent gold nanoparticles]]> https://www.researchpad.co/article/elastic_article_14337 Because lysosomes play critical roles in multiple cellular functions and are associated with many diseases, studying them at the subcellular level could elucidate their functionality and support the discovery of therapeutic drugs for treating those diseases. The commonly used dyes for super-resolution imaging of lysosomes are the commercial molecular LysoTrackers. But the tolerance to changes in the lysosomal microenvironment and to lysosomal membrane permeabilization (LMP) and the photostability of the LysoTrackers are worrisome. The purpose of our study was to evaluate the feasibility of performing a fluorescent gold nanoprobe for super-resolution observation of lysosomal dynamics in living cells and compare it to the commercial LysoTrackers.

Methods: The nanoprobe Cy5@Au NP contained three parts: a bio-inert gold core, a biocompatible polyethylene glycol spacer, and a fluorophore cyanine 5. Structured illumination microscopy (SIM) was employed to capture the fluorescence of Cy5@Au NPs in cells. The tolerance assays to changes in the lysosomal microenvironment and to LMP, the photobleaching assay, and the long-term lysosomes labelling assay of Cy5@Au NPs were compared with commercial LysoTrackers. The super-resolution observation of lysosomal dynamics with Cy5@Au NPs was performed.

Results: Cy5@Au NPs can light up lysosomes specifically under SIM. Compared with commercial lysosomal molecular probes, Cy5@Au NPs exhibited stronger tolerance in lysosomes during various treatments, and changes in the lysosomal microenvironment and LMP did not cause Cy5@Au NPs to lose track of their targets. Cy5@Au NPs demonstrated an excellent anti-photobleaching ability, and a long-term labelling assay revealed that they could label lysosomes more than 3 d. Biological events of lysosomes such as the kiss-and-run process, fusion, fission, and mitophagy were recorded with the fluorescent Cy5@Au NPs under SIM.

Conclusions: The nanoprobe Cy5@Au NP was successfully used as a lysosomal probe for the super-resolution observation in living cells and found to overcome the limitations of commercial LysoTrackers. Our results thus confirm that nanoparticles can be useful tools for subcellular super-resolution imaging and highlight new avenues for using nanoparticles in biology.

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<![CDATA[Investigation of the role and mechanism of ARHGAP5-mediated colorectal cancer metastasis]]> https://www.researchpad.co/article/elastic_article_14334 Background: Metastatic colorectal cancer (CRC) is a lethal disease; however, the underlying molecular mechanisms remain unclear and require further study.

Methods: RNA-Seq, PCR, Western blotting, immunohistochemistry, ChIP and RNAi assays were performed to investigate Rho GTPase-activating protein 5 (ARHGAP5, aslo known as p190RhoGAP-B, p190-B) expression and the clinical relevance, functional roles and regulatory mechanisms of this protein using human CRC cells and tissues. In vivo, two cell-based xenograft models were used to evaluate the roles of ARHGAP5 in CRC metastasis.

Results: Here, we report that ARHGAP5 expression is significantly increased in metastatic CRC tissues and is inversely associated with patient overall survival. The suppression of ARHGAP5 reduces CRC cell metastasis in vitro and in cell-based xenograft models. Furthermore, we show that ARHGAP5 promotes CRC cell epithelial-mesenchymal transition by negatively regulating RhoA activity. Mechanistically, cAMP response element-binding protein (CREB1) transcriptionally upregulates ARHGAP5 expression, and decreased miR-137 further contributes to ARHGAP5 mRNA stability in CRC.

Conclusions: Overall, our study highlights the crucial function of ARHGAP5 in CRC metastasis, thus suggesting novel prognostic biomarkers and hypothetical therapeutic targets.

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<![CDATA[A novel vaccine candidate based on chimeric virus-like particle displaying multiple conserved epitope peptides induced neutralizing antibodies against EBV infection]]> https://www.researchpad.co/article/elastic_article_14330 Rationale: Epstein-Barr virus (EBV) is the causative pathogen for infectious mononucleosis and many kinds of malignancies including several lymphomas such as Hodgkin's lymphoma, Burkitt's lymphoma and NK/T cell lymphoma as well as carcinomas such as nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBV-GC). However, to date no available prophylactic vaccine was launched to the market for clinical use.

Methods: To develop a novel vaccine candidate to prevent EBV infection and diseases, we designed chimeric virus-like particles (VLPs) based on the hepatitis B core antigen (HBc149). Various VLPs were engineered to present combinations of three peptides derived from the receptor binding domain of EBV gp350. All the chimeric virus-like particles were injected into Balb/C mice for immunogenicity evaluation. Neutralizing titer of mice sera were detected using an in vitro cell model.

Results: All chimeric HBc149 proteins self-assembled into VLPs with gp350 epitopes displayed on the surface of spherical particles. Interestingly, the different orders of the three epitopes in the chimeric proteins induced different immune responses in mice. Two constructs (149-3A and 149-3B) induced high serum titer against the receptor-binding domain of gp350. Most importantly, these two VLPs elicited neutralizing antibodies in immunized mice, which efficiently blocked EBV infection in cell culture. Competition analysis showed that sera from these mice contained antibodies to a major neutralizing epitope recognized by the strong neutralizing mAb 72A1.

Conclusion: Our data demonstrate that HBc149 chimeric VLPs provide a valuable platform to present EBV gp350 antigens and offer a robust basis for the development of peptide-based candidate vaccines against EBV.

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<![CDATA[Quantitative chemical imaging of breast calcifications in association with neoplastic processes]]> https://www.researchpad.co/article/elastic_article_14328 Calcifications play an essential role in early breast cancer detection and diagnosis. However, information regarding the chemical composition of calcifications identified on mammography and histology is limited. Detailed spectroscopy reveals an association between the chemical composition of calcifications and breast cancer, warranting the development of novel analytical tools to better define calcification types. Previous investigations average calcification composition across broad tissue sections with no spatially resolved information or provide qualitative visualization, which prevents a robust linking of specific spatially resolved changes in calcification chemistry with the pathologic process.

Method: To visualize breast calcification chemical composition at high spatial resolution, we apply hyperspectral stimulated Raman scattering (SRS) microscopy to study breast calcifications associated with a spectrum of breast changes ranging from benign to neoplastic processes, including atypical ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma. The carbonate content of individual breast calcifications is quantified using a simple ratiometric analysis.

Results: Our findings reveal that intra-sample calcification carbonate content is closely associated with local pathological processes. Single calcification analysis supports previous studies demonstrating decreasing average carbonate level with increasing malignant potential. Sensitivity and specificity reach >85% when carbonate content level is used as the single differentiator in separating benign from neoplastic processes. However, the average carbonate content is limiting when trying to separate specific diagnostic categories, such as fibroadenoma and invasive ductal carcinoma. Second harmonic generation (SHG) data can provide critical information to bridge this gap.

Conclusion: SRS, combined with SHG, can be a valuable tool in better understanding calcifications in carcinogenesis, diagnosis, and possible prognosis. This study not only reveals previously unknown large variations of breast microcalcifications in association with local malignancy but also corroborates the clinical value of linking microcalcification chemistry to breast malignancy. More importantly, it represents an important step in the development of a label-free imaging strategy for breast cancer diagnosis with tremendous potential to address major challenges in diagnostic discordance in pathology.

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<![CDATA[Faster, sharper, more precise: Automated Cluster-FLIM in preclinical testing directly identifies the intracellular fate of theranostics in live cells and tissue]]> https://www.researchpad.co/article/elastic_article_14317 Fluorescence microscopy is widely used for high content screening in 2D cell cultures and 3D models. In particular, 3D tissue models are gaining major relevance in modern drug development. Enabling direct multiparametric evaluation of complex samples, fluorescence lifetime imaging (FLIM) adds a further level to intensity imaging by the sensitivity of the fluorescence lifetime to the microenvironment. However, the use of FLIM is limited amongst others by the acquisition of sufficient photon numbers without phototoxic effects in live cells. Herein, we developed a new cluster-based analysis method to enhance insight, and significantly speed up analysis and measurement time for the accurate translation of fluorescence lifetime information into pharmacological pathways.

Methods: We applied a fluorescently-labeled dendritic core-multishell nanocarrier and its cargo Bodipy as molecules of interest (MOI) to human cells and reconstructed human tissue. Following the sensitivity and specificity assessment of the fitting-free Cluster-FLIM analysis of data in silico and in vitro, we evaluated the dynamics of cellular molecule uptake and intracellular interactions. For 3D live tissue investigations, we applied multiphoton (mp) FLIM. Owing to Cluster-FLIM's statistics-based fitting-free analysis, we utilized this approach for automatization.

Results: To discriminate the fluorescence lifetime signatures of 5 different fluorescence species in a single color channel, the Cluster-FLIM method requires only 170, respectively, 90 counts per pixel to obtain 95% sensitivity (hit rate) and 95% specificity (correct rejection rate). Cluster-FLIM revealed cellular interactions of MOIs, representing their spatiotemporal intracellular fate. In a setting of an automated workflow, the assessment of lysosomal trapping of the MOI revealed relevant differences between normal and tumor cells, as well as between 2D and 3D models.

Conclusion: The automated Cluster-FLIM tool is fitting-free, providing images with enhanced information, contrast, and spatial resolution at short exposure times and low fluorophore concentrations. Thereby, Cluster-FLIM increases the applicability of FLIM in high content analysis of target molecules in drug development and beyond.

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<![CDATA[SUMO1 modification of methyltransferase-like 3 promotes tumor progression via regulating Snail mRNA homeostasis in hepatocellular carcinoma]]> https://www.researchpad.co/article/elastic_article_14316 Rationale: Hepatocellular carcinoma (HCC) is one of the leading causes of mortality worldwide. Methyltransferase-like 3 (Mettl3), an RNA N6-methyladenosine (m6A) methyltransferase, has been shown to act as an oncogene in several human cancers. However, the regulatory role of posttranslational modifications of Mettl3 in liver cancer remains elusive.

Methods: SUMOylation was analyzed using immunoprecipitation and western blot assays. In vitro and in vivo biological functions were examined using MTS, colony formation, wound healing, transwell, apoptosis, and viability assays and the BALB/c nude mouse model, respectively. Immunohistochemistry was conducted to evaluate the prognostic value of Mettl3 expression in HCC. The regulatory mechanism of Mettl3 in HCC was investigated by m6A dot blot, immunofluorescence, dual luciferase reporter, protein stability, and RNA stability assays.

Results: Mettl3 was found to be SUMOylated by a small ubiquitin-like modifier SUMO1. Further, SUMOylation of Mettl3 was increased upon mitogen stimulation, which correlated with UBC9 upregulation, and was positively correlated with high metastatic potential of liver cancer. Finally, SUMOylation of Mettl3 was found to regulate HCC progression via controlling Snail mRNA homeostasis in an m6A methyltransferase activity-dependent manner.

Conclusions: This study revealed a novel mechanism of SUMOylated Mettl3-mediated Snail mRNA homeostasis, identifying the UBC9/SUMOylated Mettl3/Snail axis as a novel mediator of the SUMO pathway involved in HCC progression.

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<![CDATA[Hepatoma cell-intrinsic TLR9 activation induces immune escape through PD-L1 upregulation in hepatocellular carcinoma]]> https://www.researchpad.co/article/elastic_article_14315 A TLR9 agonist in combination with a PD-1 inhibitor produced powerful antitumor responses in a clinical trial despite TLR9 agonists as monotherapies failing to generate systemic antitumor immune responses due to immunosuppressive effects. However, the mechanism involved in the improved response induced by their combination remains unknown.

Methods: Subcutaneous and orthotopic Hepa1-6 tumor model was used for single-drug and combined-drug treatment. We used TLR9 agonist stimulation or lentiviral vectors to overexpress TLR9 and activate TLR9 signaling. We next investigated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation mediated by TLR9. Tissue chips were used to analyze the relationships among TLR9, PARP1, p-STAT3 and PD-L1 expression.

Results: In this study, we found that the TLR9 agonist in combination with anti-PD-1 therapy or anti-PD-L1 therapy yielded an additive effect that inhibited HCC growth in mice. Mechanistically, we found that TLR9 promoted PD-L1 transcription by enhancing STAT3 Tyr705 phosphorylation. Then, we observed that TLR9 negatively regulated PARP1 expression, which mediated a decrease in STAT3 PARylation and an increase in STAT3 Tyr705 phosphorylation. Moreover, we found that TLR9 enhanced PARP1 autoPARylation by inhibiting PARG expression, which then promoted the RNF146-mediated ubiquitination and subsequent degradation of PARP1. Finally, we observed positive associations between TLR9 and p-STAT3 (Tyr705) or PD-L1 expression and negative associations between TLR9 and PARP1 in HCC patient samples.

Conclusions: We showed that hepatoma cell-intrinsic TLR9 activation regulated the crosstalk between PARP1 autoPARylation and ubiquitination and between STAT3 PARylation and phosphorylation, which together upregulated PD-L1 expression and finally induces immune escape. Therefore, combination therapy with a TLR9 agonist and an anti-PD-1 antibody or anti-PD-L1 had much better antitumor efficacy than either monotherapy in HCC.

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<![CDATA[<i>In vivo</i> imaging of Zika virus reveals dynamics of viral invasion in immune-sheltered tissues and vertical propagation during pregnancy]]> https://www.researchpad.co/article/elastic_article_14314 Rationale: Zika virus (ZIKV) is a pathogenic virus known to cause a wide range of congenital abnormalities, including microcephaly, Guillain-Barre syndrome, meningoencephalitis, and other neurological complications, in humans. This study investigated the noninvasive detection of ZIKV infection in vivo, which is necessary for elucidating the virus's mechanisms of viral replication and pathogenesis, as well as to accelerate the development of anti-ZIKV therapeutic strategies.

Methods: In this study, a recombinant ZIKV harbouring Nluc gene (ZIKV-Nluc) was designed, recovered, and purified. The levels of bioluminescence were directly correlated with viral loads in vitro and in vivo. The dynamics of ZIKV infection in A129 (interferon (IFN)-α/β receptor deficient), AG6 (IFN-α/β and IFN-γ receptor deficient), and C57BL/6 mice were characterized. Pregnant dams were infected with ZIKV-Nluc at E10 via intra footpad injection. Then, the pooled immune sera (anti-ZIKV neutralizing antibodies) #22-1 in ZIKV-Nluc virus-infected mice were visualized.

Results: ZIKV-Nluc showed a high genetic stability and replicated well in cells with similar properties to the wild-type ZIKV (ZIKVwt). Striking bioluminescence signals were consistently observed in animal organs, including spleen, intestine, testis, uterus/ovary, and kidney. The ileocecal junction was found to be the crucial visceral target. Infection of pregnant dams with ZIKV-Nluc showed that ZIKV was capable of crossing the maternal-fetal barrier to infect the fetuses via vertical transmission. Furthermore, it was visualized that treatment with the pooled immune sera was found to greatly restrict the spread of the ZIKV-Nluc virus in mice.

Conclusions: This study is the first to report the real-time noninvasive tracking of the progression of ZIKV invading immune-sheltered tissues and propagating vertically during pregnancy. The results demonstrate that ZIKV-Nluc represents a powerful tool for the study of the replication, dissemination, pathogenesis, and treatment of ZIKV in vitro and in vivo.

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<![CDATA[Immunohistochemical PSMA expression patterns of primary prostate cancer tissue are associated with the detection rate of biochemical recurrence with <sup>68</sup>Ga-PSMA-11-PET]]> https://www.researchpad.co/article/elastic_article_14312 Prostate-specific membrane antigen (PSMA) targeted PET has a high detection rate for biochemical recurrence (BCR) of prostate cancer (PCa). Nevertheless, even at high prostate-specific antigen (PSA) levels (> 3 ng/ml), a relevant number of PSMA-PET scans are negative, mainly due to PSMA-negative PCa. Our objective was to investigate whether PSMA-expression patterns of the primary tumour on immunohistochemistry (IHC) are associated with PSMA-PET detection rate of recurrent PCa.

Methods: Retrospective institutional review board approved single-centre analysis of patients who had undergone 68Ga-PSMA-11-PET for BCR after radical prostatectomy (RPE) between 04/2016 and 07/2019, with tumour specimens available for PSMA-IHC. Clinical information (age, PSA-level, ongoing androgen deprivation therapy (ADT), Gleason score) and PSMA-IHC of the primary tumour were collected and their relationship to results from PSMA-PET (positive/negative) was investigated using a multiple logistic regression analysis.

Results: 120 PSMA-PET scans in 74 patients were available for this analysis. Overall detection rate was 62% (74/120 scans), with a mean PSA value at scan time of 0.99 ng/ml (IQR 0.32-4.27). Of the clinical factors, only PSA-level and ADT were associated with PSMA-PET positivity. The percentage of PSMA-negative tumour area on IHC (PSMA%neg) had a significant association to PSMA-PET negativity (OR = 2.88, p < 0.001), while membranous PSMA-expression showed no association (p = 0.73). The positive predictive value of PSMA%neg ≥ 50% for a negative PSMA-PET was 85% (13/11) and for a PSMA%neg of 80% or more, 100% (9/9).

Conclusions: PSMA-negative tumour area on IHC exhibited the strongest association with negative PSMA-PET scans, beside PSA-level and ADT. Even at very high PSA levels, PSMA-PET scans were negative in most of the patients with PSMA%neg ≥ 50%.

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<![CDATA[DR-region of Na<sup>+</sup>/K<sup>+</sup>-ATPase is a target to ameliorate hepatic insulin resistance in obese diabetic mice]]> https://www.researchpad.co/article/elastic_article_14310 Reduced hepatic Na+/K+-ATPase (NKA) activity and NKAα1 expression are engaged in the pathologies of metabolism diseases. The present study was designed to investigate the potential roles of NKAα1 in hepatic gluconeogenesis and glycogenesis in both hepatocytes and obese diabetic mice.

Methods: Insulin resistance was mimicked by glucosamine (GlcN) in either human hepatocellular carcinoma (HepG2) cells or primary mouse primary hepatocytes. Obese diabetic mice were induced by high-fat diet (HFD) feeding for 12 weeks.

Results: We found that both NKA activity and NKAα1 protein level were downregulated in GlcN-treated hepatocytes and in the livers of obese diabetic mice. Pharmacological inhibition of NKA with ouabain worsened, while activation of NKAα1 with an antibody against an extracellular DR region of NKAα1 subunit (DR-Ab) prevented GlcN-induced increase in gluconeogenesis and decrease in glycogenesis. Likewise, the above results were also corroborated by the opposite effects of genetic knockout/overexpression of NKAα1 on both gluconeogenesis and glycogenesis. In obese diabetic mice, hepatic activation or overexpression of NKAα1 stimulated the PI3K/Akt pathway to suppress hyperglycemia and improve insulin resistance. More importantly, loss of NKA activities in NKAα1+/- mice was associated with more susceptibility to insulin resistance following HFD feeding.

Conclusions: Our findings suggest that NKAα1 is a physiological regulator of glucose homoeostasis and its DR-region is a novel target to treat hepatic insulin resistance.

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