Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Author Correction: High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

In Fig. 4e of this Article, the labels for 'Control' and 'HFD' were reversed ('Control' should have been labelled blue rather than purple, and 'HFD' should have been labelled purple rather than blue). Similarly, in Fig. 4f of this Article, the labels for 'V' and 'GW' were reversed ('V' should have been labelled blue rather than purple, and 'GW' should have been labelled purple instead of blue). The original figure has been corrected online.

High-fat diet enhances stemness and tumorigenicity of intestinal progenitors.

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we show that high-fat diet (HFD)-induced obesity augments the numbers and function of Lgr5(+) intestinal stem cells of the mammalian intestine. Mechanistically, a HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-δ) signature in intestinal stem cells and progenitor cells (non-intestinal stem cells), and pharmacological activation of PPAR-δ recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-δ-dependent manner. Notably, HFD- and agonist-activated PPAR-δ signalling endow organoid-initiating capacity to progenitors, and enforced PPAR-δ signalling permits these progenitors to form in vivo tumours after loss of the tumour suppressor Apc. These findings highlight how diet-modulated PPAR-δ activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumours.

Diagnostic predictive values for sport-related concussions: a systematic review and diagnostic meta-analysis.

OBJECTIVE: Sport-related concussions (SRCs) can cause significant neurological symptoms, and approximately 10%-15% of athletes with SRC experience a prolonged recovery. Given the lack of visible injury on brain imaging and their varied presentations, concussions can be difficult to diagnose. A variety of tests and examination methods have been used to elicit a concussion diagnosis; however, the sensitivity and specificity of these tests are variable. The authors performed a systematic review and meta-analysis to evaluate the sensitivity and specificity of standardized tests and visible signs like balance and vision changes in the diagnosis of SRC. METHODS: A PRISMA-adherent systematic review of concussion diagnostic examinations was performed using the PubMed, MEDLINE, Scopus, Cochrane, Web of Science, and Google Scholar databases on December 1, 2022. Search terms included "concussion," "traumatic brain injury," "diagnosis," "sensitivity," and "specificity." Each method of examination was categorized into larger group-based symptomatologic presentations or standardized tools. The primary outcome was the diagnosis of concussion. Pooled specificity and sensitivity for each method were calculated using a meta-analysis of proportion and were hierarchically ranked using P-scores calculated from a diagnostic frequentist network meta-analysis. RESULTS: Thirty full-length articles were identified for inclusion, 13 of which evaluated grouped symptomology examinations (balance and overall clinical presentation) and 17 of which evaluated established formalized tools (ImPACT, King-Devick [K-D] Test, Sport Concussion Assessment Tool [SCAT]). The pooled specificity of the examination methods differed minimally (0.8-0.85), whereas the sensitivity varied to a larger degree (0.5-0.88). In a random effects model, the SCAT had the greatest diagnostic yield (diagnostic OR 31.65, 95% CI 11.06-90.57). Additionally, P-score hierarchical ranking revealed SCAT as having the greatest diagnostic utility (p = 0.9733), followed sequentially by ImPACT, clinical presentation, K-D, and balance. CONCLUSIONS: In deciphering which concussion symptom-focused examinations and standardized tools are most accurate in making a concussion diagnosis, the authors found that the SCAT examination has the greatest diagnostic yield, followed by ImPACT, clinical presentation, and K-D, which have comparable value for diagnosis. Given the indirect nature of this analysis, however, further comparative studies are needed to validate the findings.

Automatic reagent handling and assay processing of human biospecimens inside a transportation container for a medical disaster response against radiation.

Biological materials can be shipped off-site for diagnostic, therapeutic and research purposes. They usually are kept in certain environments for their final application during transportation. However, active reagent handling during transportation from a collection site to a laboratory or biorepository has not been reported yet. In this paper, we show the application of a micro-controlled centrifugal microfluidic system inside a shipping container that can add reagent to an actively cultured human blood sample during transportation to ensure a rapid biodosimetry of cytokinesis-block micronucleus (CBMN) assay. The newly demonstrated concept could have a significant impact on rapid biodosimetry triage for medical countermeasure in a radiological disaster. It also opens a new capability in accelerated sample processing during transportation for biomedical and healthcare applications.

Reinforcement regulates timing variability in thalamus.

Learning reduces variability but variability can facilitate learning. This paradoxical relationship has made it challenging to tease apart sources of variability that degrade performance from those that improve it. We tackled this question in a context-dependent timing task requiring humans and monkeys to flexibly produce different time intervals with different effectors. We identified two opposing factors contributing to timing variability: slow memory fluctuation that degrades performance and reward-dependent exploratory behavior that improves performance. Signatures of these opposing factors were evident across populations of neurons in the dorsomedial frontal cortex (DMFC), DMFC-projecting neurons in the ventrolateral thalamus, and putative target of DMFC in the caudate. However, only in the thalamus were the performance-optimizing regulation of variability aligned to the slow performance-degrading memory fluctuations. These findings reveal how variability caused by exploratory behavior might help to mitigate other undesirable sources of variability and highlight a potential role for thalamocortical projections in this process.

Colonoscopy-based colorectal cancer modeling in mice with CRISPR-Cas9 genome editing and organoid transplantation.

Most genetically engineered mouse models (GEMMs) of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations-such as the subcutaneous space, kidney capsule, or cecal wall-that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection. These techniques can be adapted to deliver viral vectors carrying Cre recombinase, CRISPR-Cas9 components, CRISPR-engineered mouse tumor organoids, or human cancer organoids to mice to model the adenoma-carcinoma-metastasis sequence of tumor progression. The colonoscopy injection procedure takes ∼15 min, including preparation. In our experience, anyone with reasonable hand-eye coordination can become proficient with mouse colonoscopy and mucosal injection with a few hours of practice. These approaches are ideal for a wide range of applications, including assessment of gene function in tumorigenesis, examination of tumor-stroma interactions, studies of cancer metastasis, and translational research with patient-derived cancers.

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;KrasG12D/+;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.

Corrigendum: In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

This corrects the article DOI: 10.1038/nbt.3836.

A handy review of carpal tunnel syndrome: From anatomy to diagnosis and treatment.

Carpal tunnel syndrome (CTS) is the most commonly diagnosed disabling condition of the upper extremities. It is the most commonly known and prevalent type of peripheral entrapment neuropathy that accounts for about 90% of all entrapment neuropathies. This review aims to provide an outline of CTS by considering anatomy, pathophysiology, clinical manifestation, diagnostic modalities and management of this common condition, with an emphasis on the diagnostic imaging evaluation.