A Systematic Review Examining the Experimental Methodology Behind In Vivo Testing of Hiatus Hernia and Diaphragmatic Hernia Mesh

Introduction Mesh implants are regularly used to help repair both hiatus hernias (HH) and diaphragmatic hernias (DH). In vivo studies are used to test not only mesh safety, but increasingly comparative efficacy. Our work examines the field of in vivo mesh testing for HH and DH models to establish current practices and standards. Method This systematic review was registered with PROSPERO. Medline and Embase databases were searched for relevant in vivo studies. Forty-four articles were identified and underwent abstract review, where 22 were excluded. Four further studies were excluded after full-text review—leaving 18 to undergo data extraction. Results Of 18 studies identified, 9 used an in vivo HH model and 9 a DH model. Five studies undertook mechanical testing on tissue samples—all uniaxial in nature. Testing strip widths ranged from 1–20 mm (median 3 mm). Testing speeds varied from 1.5–60 mm/minute. Upon histology, the most commonly assessed structural and cellular factors were neovascularisation and macrophages respectively (n = 9 each). Structural analysis was mostly qualitative, where cellular analysis was equally likely to be quantitative. Eleven studies assessed adhesion formation, of which 8 used one of four scoring systems. Eight studies measured mesh shrinkage. Discussion In vivo studies assessing mesh for HH and DH repair are uncommon. Within this relatively young field, we encourage surgical and materials testing institutions to discuss its standardisation.

USP1 Promotes GC Metastasis via Stabilizing ID2

Gastric cancer (GC) is one of the most common malignant tumors all over the world. And recurrence and metastasis are still the main causes of low survival rate for advanced GC. USP1 has been shown overexpressed in multiple cancers, which indicate its important biomarker in tumorigenesis and development. Our study is aimed at defining the exact role of USP1 on GC metastasis and the underlying mechanism. USP1 was firstly found overexpressed in GC tissues and relatively high-expression levels conferred poor survival rates. Then, real-time cellular analysis (RTCA) showed that USP1 knockdown inhibited GC metastasis both in vitro and in vivo. Mechanically, we demonstrated that USP1 promoted GC metastasis via upregulating ID2 expression and further confirmed that USP1 stabilized ID2 expression through deubiquitinating ID2 in GC. In conclusion, our study showed that USP1 promoted GC metastasis via stabilizing ID2 expression, which provides a potential biomarker and therapy target for GC.

PrPC as a Transducer of Physiological and Pathological Signals

After the discovery of prion phenomenon, the physiological role of the cellular prion protein (PrPC) remained elusive. In the past decades, molecular and cellular analysis has shed some light regarding interactions and functions of PrPC in health and disease. PrPC, which is located mainly at the plasma membrane of neuronal cells attached by a glycosylphosphatidylinositol (GPI) anchor, can act as a receptor or transducer from external signaling. Although the precise role of PrPC remains elusive, a variety of functions have been proposed for this protein, namely, neuronal excitability and viability. Although many issues must be solved to clearly define the role of PrPC, its connection to the central nervous system (CNS) and to several misfolding-associated diseases makes PrPC an interesting pharmacological target. In a physiological context, several reports have proposed that PrPC modulates synaptic transmission, interacting with various proteins, namely, ion pumps, channels, and metabotropic receptors. PrPC has also been implicated in the pathophysiological cell signaling induced by β-amyloid peptide that leads to synaptic dysfunction in the context of Alzheimer’s disease (AD), as a mediator of Aβ-induced cell toxicity. Additionally, it has been implicated in other proteinopathies as well. In this review, we aimed to analyze the role of PrPC as a transducer of physiological and pathological signaling.

Multiple alterations in glutamatergic transmission and dopamine D2 receptor splicing in induced pluripotent stem cell-derived neurons from patients with familial schizophrenia

An increasing body of evidence suggests that impaired synapse development and function are associated with schizophrenia; however, the underlying molecular pathophysiological mechanism of the disease remains largely unclear. We conducted a family-based study combined with molecular and cellular analysis using induced pluripotent stem cell (iPSC) technology. We generated iPSCs from patients with familial schizophrenia, differentiated these cells into neurons, and investigated the molecular and cellular phenotypes of the patient’s neurons. We identified multiple altered synaptic functions, including increased glutamatergic synaptic transmission, higher synaptic density, and altered splicing of dopamine D2 receptor mRNA in iPSC-derived neurons from patients. We also identified patients’ specific genetic mutations using whole-exome sequencing. Our findings support the notion that altered synaptic function may underlie the molecular and cellular pathophysiology of schizophrenia, and that multiple genetic factors cooperatively contribute to the development of schizophrenia.

B and T Cell Responses after a Third Dose of SARS-CoV-2 Vaccine in Kidney Transplant Recipients

Background: Accumulating evidence suggests that solid organ transplant recipients, as opposed to the general population, show strongly impaired responsiveness towards standard SARS-CoV-2 mRNA-based vaccination, demanding alternative strategies for protection of this vulnerable group. Methods: In line with recent recommendations, a third dose of either heterologous ChAdOx1 (AstraZeneca) or homologous BNT162b2 (BioNTech) was administered to 25 kidney transplant recipients (KTR) without humoral response after 2 doses of BNT162b2, followed by analysis of serological responses and vaccine-specific B- and T-cell immunity. Results: 9/25 (36%) KTR under standard immunosuppressive treatment seroconverted until day 27 after the third vaccination, while one patient developed severe COVID-19 infection immediately after vaccination. Cellular analysis seven days after the third dose showed significantly elevated frequencies of viral spike protein receptor binding domain specific B cells in humoral responders as compared to non-responders. Likewise, portions of spike-reactive CD4+ T helper cells were significantly elevated in seroconverting patients. Furthermore, overall frequencies of IL-2+, IL-4+ and polyfunctional CD4+ T cells significantly increased after the third dose, whereas memory/effector differentiation remained unaffected. Conclusions: Our data suggest that a fraction of transplant recipients benefits from triple vaccination, where seroconversion is associated with quantitative and qualitative changes of cellular immunity. At the same time, the study highlights that modified vaccination approaches for immunosuppressed patients still remain an urgent medical need.

Flow cytometry and pharmacokinetics

Multiparametric flow cytometry is a powerful cellular analysis tool used in various stages of drug development. In adoptive cell therapies, the flow cytometry methods are used for the evaluation of advanced cellular products during manufacturing and to monitor cellular kinetics after infusion. In this report, we discussed the bioanalytical method development challenges to monitor cellular kinetics in CAR-T cell therapies. These method development challenges include procuring positive control samples for the development of the method, flow cytometry panel design, LLOQ, prestain sample stability, staining reagents and data analysis.

Comparative cellular analysis of motor cortex in human, marmoset and mouse

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch–seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Performance evaluation of automated cell counts compared with reference methods for body fluid analysis

Objectives Cellular analysis of body fluids (BFs) can assist clinicians for the diagnosis of many medical conditions. The aim of this work is the evaluation of the analytical performance of the UF-5000 body fluid mode (UF-BF) analyzer compared to the gold standard method (optical microscopy, OM) and to XN-1000 (XN-BF), another analyzer produced by the same manufacturer (Sysmex) and with a similar technology for BF analysis. Methods One hundred BF samples collected in K3EDTA tubes were analyzed by UF-BF, XN-BF and OM. The agreement was evaluated using Passing and Bablok regression and Bland–Altman plot analysis. The receiver operating characteristic (ROC) curves were selected for evaluating the diagnostic agreement between OM classification and UF-BF parameters. Results Comparison between UF-BF and OM, in all BF types, showed Passing and Bablok’s slope comprised between 0.99 (polymorphonuclear cells count, PMN-BF) and 1.39 (mononuclear cells count, MN-BF), the intercepts ranged between 26.47 (PMN-BF parameter) and 226.80 (white blood cell count). Bland–Altman bias was comprised between 7.3% (total cell count, TC-BF) and 52.9% (MN-BF). Comparison between UF-BF and XN-BF in all BF showed slopes ranged between 1.07 (TC-BF and PMN-BF) and 1.16 (MN-BF), intercepts ranged between 8.30 (PMN) and 64.78 (WBC-BF). Bland–Altman bias ranged between 5.8 (TC-BF) and 21.1% (MN-BF). The ROC curve analysis showed an area under the curve ranged between 0.9664 and 1.000. Conclusions UF-BF shows very good performance for the differential counts of cells in ascitic, pleural and synovial fluids and therefore it is useful to screen and count cells in this type of BF.