Nuclear mechanics is remodeled not only by extracellular forces but also by internal modifications, such as those induced by viral infections. During herpes simplex virus type 1 infection, the nuclear structures undergo drastic reorganization, but little is known about how nuclear mechanobiology changes as a result. We show that the nucleus softens dramatically during the infection. To understand the phenomenon, we used advanced microscopy and computational modeling. We discovered that the enlarged viral replication compartment had a low biomolecular density, partially explaining the observed nuclear softening. The mobility of the nuclear lamina decreased, which suggests increased rigidity and an inability to induce softening. However, computational modeling supported by experimental data showed that reduced outward forces, such as cytoskeletal pull and intranuclear osmotic pressure acting both on and within the nucleus, can explain the decreased nuclear stiffness. Our findings reveal that during infection, the nucleus is subject to changes in multiple mechanical forces, leading to decreased nuclear stiffness.

During infection, Herpes simplex virus type 1 (HSV-1) alters the mitochondrial structure and function of the host cell. Live-cell imaging with fluorescent reporters revealed increased mitochondrial calcium and a transient ROS enrichment after HSV-1 infection. Notably, cells co-transfected with a calcium reporter displayed smaller viral replication compartments, while those with a ROS reporter exhibited average growth of viral replication compartments. Our findings suggest that the virus-induced increase in mitochondrial calcium, followed by an increased amount of bound calcium reporter, interferes with the progression of the infection.

Viruses target mitochondria to promote their replication, and infection-induced stress during the progression of infection leads to the regulation of antiviral defenses and mitochondrial metabolism which are opposed by counteracting viral factors. The precise structural and functional changes that underlie how mitochondria react to the infection remain largely unclear. Here we show extensive transcriptional remodeling of protein-encoding host genes involved in the respiratory chain, apoptosis, and structural organization of mitochondria as herpes simplex virus type 1 lytic infection proceeds from early to late stages of infection. High-resolution microscopy and interaction analyses unveiled infection-induced emergence of rough, thin, and elongated mitochondria relocalized to the perinuclear area, a significant increase in the number and clustering of endoplasmic reticulum-mitochondria contact sites, and thickening and shortening of mitochondrial cristae. Finally, metabolic analyses demonstrated that reactivation of ATP production is accompanied by increased mitochondrial Ca2+ content and proton leakage as the infection proceeds. Overall, the significant structural and functional changes in the mitochondria triggered by the viral invasion are tightly connected to the progression of the virus infection.

Background Older patients with diabetic kidney disease (DKD) often do not receive optimal pharmacological treatment. Current clinical practice guidelines (CPGs) do not incorporate the concept of personalised care. Clinical decision support (CDS) algorithms that consider both evidence and personalised care to improve patient outcomes can improve the care of older adults. The aim of this research is to design and validate a CDS algorithm for prescribing renin-angiotensin-aldosterone system inhibitors (RAASi) for older patients with diabetes.Methods The design of the CDS tool included the following phases: (1) gathering evidence from systematic reviews and meta-analyses of randomised clinical trials to determine the number needed to treat (NNT) and time-to-benefit (TTB) values applicable to our target population for use in the algorithm. (2) Building a list of potential cases that addressed different prescribing scenarios (starting, adding or switching to RAASi). (3) Reviewing relevant guidelines and extracting all recommendations related to prescribing RAASi for DKD. (4) Matching NNT and TTB with specific clinical cases. (5) Validating the CDS algorithm using Delphi technique.Results We created a CDS algorithm that covered 15 possible scenarios and we generated 36 personalised and nine general recommendations based on the calculated and matched NNT and TTB values and considering the patient’s life expectancy and functional capacity. The algorithm was validated by experts in three rounds of Delphi study.Conclusion We designed an evidence-informed CDS algorithm that integrates considerations often overlooked in CPGs. The next steps include testing the CDS algorithm in a clinical trial.

Determining rules for gene expression regulation is an important step toward predicting how cells are decoding the genome sequence to create a wide variety of phenotypes. Recent advances in imaging technologies revealed the stochastic nature of gene expression, in which different numbers of mRNA and protein molecules can be created in cells that have the same genome sequence (Elowitz, 2002); Kaufmann and van Oudenaarden, 2007). An early research revealed that this stochasticity is yielded by two factors: intrinsic and extrinsic noise. While the former is due to instant random chemical reactions in gene expression process, the latter is caused by cell specific molecular states emerging from the integration of gene expression over a longer time (Elowitz, 2002); Kaufmann and van Oudenaarden 2007). This finding inspired studies to investigate how cells deterministically cause robust phenotypes under such stochasticity. In contrast, this also motivated investigations on how cells utilize this stochasticity to generate different kinds of phenotypes for processes such as neural development (Johnson et al., 2015), emergence of bacterial resistance (Sánchez-Romero and Casadesús, 2014), or cancer development (Marusyk et al., 2012; Junttila and de Sauvage, 2013).

Purpose of review: Diabetes affects almost a 10th of the Canadian population, and diabetic nephropathy is one of its main complications. It remains a leading cause of kidney failure despite the availability of effective treatments. Sources of information: The sources of information are iterative discussions between health care professionals and patient partners and literature collected through the search of multiple databases. Methods: Major pitfalls related to optimal diabetic nephropathy care were identified through discussions between patient partners and clinician researchers. We identified underlying factors that were common between pitfalls. We then conducted a narrative review of strategies to overcome them, with a focus on Canadian initiatives. Key findings: We identified 5 pitfalls along the diabetic nephropathy trajectory, including a delay in diabetes diagnosis, suboptimal glycemic control, delay in the detection of kidney involvement, suboptimal kidney protection, and deficient management of advanced chronic kidney disease. Several innovative care models and approaches have been proposed to address these pitfalls; however, they are not consistently applied. To improve diabetic nephropathy care in Canada, we recommend focusing initiatives on improving awareness of diabetic nephropathy, improving access to timely evidence-based care, fostering inclusive patient-centered care environment, and generating new evidence that supports complex disease management. It is imperative that patients and their families are included at the center of these initiatives. Limitations: This review was limited to research published in peer-reviewed journals. We did not perform a systematic review of the literature; we included articles that were relevant to the major pitfalls identified by our patient partners. Study quality was also not formally assessed. The combination of these factors limits the scope of our conclusions.

The nuclear export factor CRM1-mediated pathway is known to be important for the nuclear egress of progeny parvovirus capsids in the host cells with virus-mediated cell cycle arrest at G2/M. However, it is still unclear whether this is the only pathway by which capsids exit the nucleus. Our studies show that the nuclear egress of DNA-containing full canine parvovirus. capsids was reduced but not fully inhibited when CRM1-mediated nuclear export was prevented by leptomycin B. This suggests that canine parvovirus capsids might use additional routes for nuclear escape. This hypothesis was further supported by our findings that nuclear envelope (NE) permeability was increased at the late stages of infection. Inhibitors of cell cycle regulatory protein cyclin-dependent kinase 1 (Cdk1) and pro-apoptotic caspase 3 prevented the NE leakage. The change in NE permeability could be explained by the regulation of the G2/M checkpoint which is accompanied by early mitotic and apoptotic events. The model of G2/M checkpoint activation was supported by infection-induced nuclear accumulation of cyclin B1 and Cdk1. Both NE permeability and nuclear egress of capsids were reduced by the inhibition of Cdk1. Additional proof of checkpoint function regulation and promotion of apoptotic events was the nucleocytoplasmic redistribution of nuclear transport factors, importins, and Ran, in late infection. Consistent with our findings, post-translational histone acetylation that promotes the regulation of several genes related to cell cycle transition and arrest was detected. In conclusion, the model we propose implies that parvoviral capsid egress partially depends on infection-induced G2/M checkpoint regulation involving early mitotic and apoptotic events.

Background The cause of podocyte injury in idiopathic nephrotic syndrome (INS) remains unknown. Although recent evidence points to the role of B cells and autoimmunity, the lack of animal models mediated by autoimmunity limits further research. We aimed to establish a mouse model mimicking human INS by immunizing mice with Crb2, a transmembrane protein expressed at the podocyte foot process. Methods C3H/HeN mice were immunized with the recombinant extracellular domain of mouse Crb2. Serum anti-Crb2 antibody, urine protein-to-creatinine ratio, and kidney histology were studied. For signaling studies, a Crb2-expressing mouse podocyte line was incubated with anti-Crb2 antibody. Results Serum anti-Crb2 autoantibodies and significant proteinuria were detected 4 weeks after the first immunization. The proteinuria reached nephrotic range at 9-13 weeks and persisted up to 29 weeks. Initial kidney histology resembled minimal change disease in humans, and immunofluorescence staining showed delicate punctate IgG staining in the glomerulus, which colocalized with Crb2 at the podocyte foot process. A subset of mice developed features resembling FSGS after 18 weeks. In glomeruli of immunized mice and in Crb2-expressing podocytes incubated with anti-Crb2 antibody, phosphorylation of ezrin, which connects Crb2 to the cytoskeleton, increased, accompanied by altered Crb2 localization and actin distribution. Conclusion The results highlight the causative role of anti-Crb2 autoantibody in podocyte injury in mice. Crb2 immunization could be a useful model to study the immunologic pathogenesis of human INS, and may support the role of autoimmunity against podocyte proteins in INS.

ABSTRACT The nucleolus is a dynamic nuclear structure which plays important roles in ribosome biogenesis and cellular stress response to stressors such as viral infection. The nucleolus and nucleolar proteins are essential for the progression of infection by several viruses. Consequently, viral infection often induces alterations in nucleolar structure and composition. Here, we applied a deep learning algorithm segmentation and nucleolin labeling to analyze the nucleolar changes induced by autonomous parvovirus infection. Our results show that the size of nucleoli decreases and nucleolin is released into the nucleoplasm in late infection. Analyses of ki-67, one of the NS2-associated nucleolar proteins and a key factor in nucleolar organization, showed that the interaction between ki-67 with DNA increases in infection. The infection initiated by a viral clone lacking an intact NS2 failed to decrease the nucleolar size, however, the orientation of the nucleoli was changed. Our results suggest that parvoviruses modify and exploit nucleoli and nucleolar proteins during infection, and NS2 protein might play a role in the regulation of these processes. Authors summary Autonomous parvoviruses with small genomes and only a small number of encoded proteins depend on the cellular nuclear machinery for their efficient replication. The nucleolus is an active nuclear structure involved in the essential functions of cells including synthesis of ribosomal RNA and regulation of cell growth and cellular stress. Many viruses target the nucleoli to manipulate cellular antiviral defense and to facilitate their replication by recruiting nucleolar proteins. Capsids of dependoparvoviruses, such as AAV, are assembled in the nucleolus, however, much less is known about how autonomous parvoviruses interact with the nucleolus. Our results suggest that the progression of autonomous parvovirus infection results in structural alteration of nucleolar structure and redistribution of nucleolar proteins. Analyses of the nonstructural protein NS2 mutants in comparison to wild-type infection indicated that NS2 might play an important role in viral interactions with the nucleolus.

Chromosome instability is a hallmark of cancer and is caused by inaccurate segregation of chromosomes. One cellular structure used to avoid this fate is the kinetochore, which binds to the centromere on the chromosome. Human centromeres are poorly understood, since sequencing and analyzing repeated alpha-satellite DNA regions, which can span a few megabases at the centromere, are particularly difficult. However, recent analyses revealed that these regions are actively transcribed and that transcription levels are tightly regulated, unveiling a possible role of RNA at the centromere. In this short review, we focus on the recent discovery of the function of human centromeric RNA in the regulation and structure of the centromere, and discuss the consequences of dysregulation of centromeric RNA in cancer.

Background: Quantification of the M-type phospholipase A2 receptor antibodies (anti-PLA2R) is now an essential tool for diagnosis and management of primary membranous nephropathy (MN). Since October 2018, Hôpital Maisonneuve-Rosemont (HMR) has been designated as Quebec’s reference center for serum anti-PLA2R antibody testing by the Institut National d’Excellence en Santé et Services Sociaux (INESSS), the regulatory body on drugs and tests usage in Quebec. Objectives: To describe the 2-step method of serum qualitative and quantitative anti-PLA2R antibody testing during its first year of use in Quebec and analyze its diagnostic value in the province’s population. Design: Retrospective cohort study. Setting: Single-center academic teaching hospital in Quebec, Canada. Patients: All patients who had a serum anti-PLA2R antibody test analyzed at HMR from October 1, 2018, to October 1, 2019, were included in the study. Measurements: Serum anti-PLA2R antibodies were screened by indirect immunofluorescence tests. If results were positive or undetermined, it was followed by a quantitative enzyme-linked immunosorbent assay (ELISA) test. Both tests were based on a commercial kit developed by the same company. Methods: We calculated sensitivity, specificity, predictive value, and likelihood ratio for both tests, using kidney biopsy findings performed at HMR as the gold standard. Results: In Quebec, a total of 1690 tests were performed among 1025 patients during the study year. A small proportion of these patients (8%) were followed at HMR. Patients tested at HMR and in the rest of Quebec had similar characteristics. Test validity was only characterized for patients tested at HMR. Sensitivity and specificity were, respectively, 58% and 100% for the qualitative test, and 71% and 100% for the quantitative test. The combined net sensitivity was 42% and the net specificity 100%. The net positive and negative predictive value were 100% and 84% respectively, whereas the net negative likelihood ratio was 0.58. Limitations: As the detailed analysis was only possible in the small proportion of patients clinically followed at HMR, there is a possible selection bias. Another potential selection bias was the focus on patients who were selected to have a kidney biopsy, probably because of more severe disease, higher probability of glomerulonephritis, or lesser number of comorbidities. Given the retrospective nature of this study, there was no systematic kidney biopsy or serum PLA2R antibody testing performed. Finally, we were unable to provide detailed information on the timing between immunosuppressive therapy and anti-PLA2R results. Conclusions: Serum anti-PLA2R antibody testing was widely used in Quebec during its first year of availability. A 2-step approach, using a qualitative test first, followed by a quantitative test if the results are positive or undetermined, appears efficient to avoid useless quantitative testing in negative patients and to better characterize undetermined results on immunofluorescence. Trial registration: Due to the retrospective nature of this study, no trial registration was performed.

PURPOSE OF REVIEW:Current immunosuppressive regimens used in kidney transplantation are sometimes ineffective and carry significant risks of morbidity and mortality. Cellular therapies are a promising alternative to prolong graft survival while minimizing treatment toxicity. We review the recently published breakthrough studies using cell therapies in kidney transplantation. RECENT FINDINGS:The reviewed phase I and II trials showed that cell therapies are feasible and safe in kidney transplantation, sometimes associated with less infectious complications than traditional regimens. Regulatory T cells and macrophages were added to the induction regimen, allowing for lower immunosuppressive drug doses without higher rejection risk. Regulatory T cells are also a treatment for subclinical rejection on the 6 months biopsy. Other strategies, like bone marrow-derived mesenchymal cells, genetically modified regulatory T cells, and chimerism-based tolerance are also really promising. In addition, to improve graft tolerance, cell therapy could be used to prevent or treat viral infection after transplantation. SUMMARY:Emerging data underline that cell therapy is a feasible and safe treatment in kidney transplantation. Although the evidence points to a benefit for transplant recipients, studies with standardized protocols, representative control groups, and longer follow-up are needed to answer the question definitively and guide future research.

We report a case of minimal change disease (MCD) with severe acute kidney injury (AKI) following the first injection of the ChAdOx1 nCoV-19 (AZD1222) vaccine from Oxford-AstraZeneca against coronavirus disease 2019 (COVID-19). A 71-year-old man with a history of dyslipidemia and a baseline serum creatinine of 0.7 mg/dL presented with nephrotic syndrome, AKI, and severe hypertension 13 days after receiving the Oxford-AstraZeneca vaccine. Refractory hyperkalemia and hypervolemia with oligoanuria prompted initiation of hemodialysis. His serum albumin was 2.6 g/dL and his urinary protein-creatinine ratio was 2,321 mg/mmol. Given a high suspicion for rapidly progressive glomerulonephritis, empirical glucocorticoid treatment was initiated (3 methylprednisolone pulses followed by high-dose prednisone). A kidney biopsy showed MCD and acute tubular injury. Kidney function and proteinuria subsequently improved, and hemodialysis was discontinued 38 days after the start of therapy. This case describes de novo MCD after the Oxford-AstraZeneca vaccine. It adds to the few published case reports of MCD after the Pfizer-BioNTech COVID-19 vaccine. Further reports and studies will be needed to elucidate whether MCD is truly associated with COVID-19 vaccination.