Disruption of the c-terminal serine protease domain of Fam111a does not alter calcium homeostasis in mice.

FAM111A gene mutations cause Kenney-Caffey syndrome (KCS) and Osteocraniostenosis (OCS), conditions characterized by short stature, low serum ionized calcium (Ca2+), low parathyroid hormone (PTH), and bony abnormalities. The molecular mechanism mediating this phenotype is unknown. The c-terminal domain of FAM111A harbors all the known disease-causing variations and encodes a domain with high homology to serine proteases. However, whether this serine protease domain contributes to the maintenance of Ca2+ homeostasis is not known. We hypothesized the disruption of the serine protease domain of FAM111A would disrupt Ca2+ homeostasis. To test this hypothesis, we generated with CRISPR/Cas9, mice with a frameshift insertion (c.1450insA) or large deletion (c.1253-1464del) mutation in the Fam111a serine protease domain. Serum-ionized Ca2+ and PTH levels were not significantly different between wild type, heterozygous, or homozygous Fam111a mutant mice. Additionally, there were no significant differences in fecal or urine Ca2+ excretion, intestinal Ca2+ absorption or overall Ca2+ balance. Only female homozygous (c.1450insA), but not heterozygous mice displayed differences in bone microarchitecture and mineral density compared to wild-type animals. We conclude that frameshift mutations that disrupt the c-terminal serine protease domain do not induce a KCS or OCS phenotype in mice nor alter Ca2+ homeostasis.

Statistical method scDEED for detecting dubious 2D single-cell embeddings and optimizing t-SNE and UMAP hyperparameters.

Two-dimensional (2D) embedding methods are crucial for single-cell data visualization. Popular methods such as t-distributed stochastic neighbor embedding (t-SNE) and uniform manifold approximation and projection (UMAP) are commonly used for visualizing cell clusters; however, it is well known that t-SNE and UMAP's 2D embeddings might not reliably inform the similarities among cell clusters. Motivated by this challenge, we present a statistical method, scDEED, for detecting dubious cell embeddings output by a 2D-embedding method. By calculating a reliability score for every cell embedding based on the similarity between the cell's 2D-embedding neighbors and pre-embedding neighbors, scDEED identifies the cell embeddings with low reliability scores as dubious and those with high reliability scores as trustworthy. Moreover, by minimizing the number of dubious cell embeddings, scDEED provides intuitive guidance for optimizing the hyperparameters of an embedding method. We show the effectiveness of scDEED on multiple datasets for detecting dubious cell embeddings and optimizing the hyperparameters of t-SNE and UMAP.

A Prospective Multicenter Standard of Care Study of Outpatient Laparoscopic Sleeve Gastrectomy.

A global shift is occurring as hospital procedures move to ambulatory surgical settings. Surgeons have performed outpatient sleeve gastrectomy (SG) in bariatric surgery since 2010. However, prospective trials are needed to ensure its safety before widespread adoption. PURPOSE: The study aimed to present a comprehensive report on the prospective data collection of 30-day outcomes of outpatient primary laparoscopic SG (LSG). This trial seeks to assess whether outpatient LSG is non-inferior to hospital-based surgery in selected patients who meet the outpatient surgery criteria set by the American Society for Metabolic and Bariatric Surgery. MATERIALS AND METHODS: This study is funded by the Society of American Gastrointestinal and Endoscopic Surgeons and has been approved by the Advarra Institutional Review Board (Pro00055990). Cognizant of the necessity for a prospective approach, data collection commenced after patients underwent primary LSG procedures, spanning from August 2021 to September 2022, at six medical centers across the USA. Data centralization was facilitated through ArborMetrix. Each center has its own enhanced recovery protocols, and no attempt was made to standardize the protocols. RESULTS: The analysis included 365 patients with a mean preoperative BMI of 43.7 ± 5.7 kg/m2. Rates for 30-day complications, reoperations, readmissions, emergency department visits, and urgent care visits were low: 1.6%, .5%, .2%, .2%, and 0%, respectively. Two patients (0.5%) experienced grade IIIb complications. There were no mortalities or leaks reported. CONCLUSION: The prospective cohort study suggests that same-day discharge following LSG seems safe in highly selected patients at experienced US centers.

scDEED: a statistical method for detecting dubious 2D single-cell embeddings and optimizing t-SNE and UMAP hyperparameters.

Two-dimensional (2D) embedding methods are crucial for single-cell data visualization. Popular methods such as t-SNE and UMAP are commonly used for visualizing cell clusters; however, it is well known that t-SNE and UMAP's 2D embedding might not reliably inform the similarities among cell clusters. Motivated by this challenge, we developed a statistical method, scDEED, for detecting dubious cell embeddings output by any 2D-embedding method. By calculating a reliability score for every cell embedding, scDEED identifies the cell embeddings with low reliability scores as dubious and those with high reliability scores as trustworthy. Moreover, by minimizing the number of dubious cell embeddings, scDEED provides intuitive guidance for optimizing the hyperparameters of an embedding method. Applied to multiple scRNA-seq datasets, scDEED demonstrates its effectiveness for detecting dubious cell embeddings and optimizing the hyperparameters of t-SNE and UMAP.

Functional characterization of age-dependent p16 epimutation reveals biological drivers and therapeutic targets for colorectal cancer.

BACKGROUND: Methylation of the p16 promoter resulting in epigenetic gene silencing-known as p16 epimutation-is frequently found in human colorectal cancer and is also common in normal-appearing colonic mucosa of aging individuals. Thus, to improve clinical care of colorectal cancer (CRC) patients, we explored the role of age-related p16 epimutation in intestinal tumorigenesis. METHODS: We established a mouse model that replicates two common genetic and epigenetic events observed in human CRCs: Apc mutation and p16 epimutation. We conducted long-term survival and histological analysis of tumor development and progression. Colonic epithelial cells and tumors were collected from mice and analyzed by RNA sequencing (RNA-seq), quantitative PCR, and flow cytometry. We performed single-cell RNA sequencing (scRNA-seq) to characterize tumor-infiltrating immune cells throughout tumor progression. We tested whether anti-PD-L1 immunotherapy affects overall survival of tumor-bearing mice and whether inhibition of both epigenetic regulation and immune checkpoint is more efficacious. RESULTS: Mice carrying combined Apc mutation and p16 epimutation had significantly shortened survival and increased tumor growth compared to those with Apc mutation only. Intriguingly, colon tumors with p16 epimutation exhibited an activated interferon pathway, increased expression of programmed death-ligand 1 (Pdl1), and enhanced infiltration of immune cells. scRNA-seq further revealed the presence of Foxp3+ Tregs and γδT17 cells, which contribute to an immunosuppressive tumor microenvironment (TME). Furthermore, we showed that a combined therapy using an inhibitor of DNA methylation and a PD-L1 immune checkpoint inhibitor is more effective for improving survival in tumor-bearing mice than blockade of either pathway alone. CONCLUSIONS: Our study demonstrated that age-dependent p16 epimutation creates a permissive microenvironment for malignant transformation of polyps to colon cancer. Our findings provide a mechanistic rationale for future targeted therapy in patients with p16 epimutation.

Humoral and T-cell-mediated responses to a pre-clinical Zika vaccine candidate that utilizes a unique insect-specific flavivirus platform.

Vaccination is critical for the control and prevention of viral outbreaks, yet conventional vaccine platforms may involve trade-offs between immunogenicity and safety. Insect-specific viruses have emerged as a novel vaccine platform to overcome this challenge. Detailed studies of humoral and T-cell responses induced by new insect-specific flavivirus (ISFV)-based vaccine platforms are needed to better understand correlates of protection and improve vaccine efficacy. Previously, we used a novel ISFV called Aripo virus (ARPV) to create a Zika virus (ZIKV) vaccine candidate (designated ARPV/ZIKV). ARPV/ZIKV demonstrated exceptional safety and single-dose efficacy, completely protecting mice from a lethal ZIKV challenge. Here, we explore the development of immune responses induced by ARPV/ZIKV immunization and evaluate its correlates of protection. Passive transfer of ARPV/ZIKV-induced immune sera to naïve mice prior to challenge emphasized the importance of neutralizing antibodies as a correlate of protection. Depletion of T-cells in vaccinated mice and adoptive transfer of ARPV/ZIKV-primed T-cells to naïve mice prior to challenge indicated that ARPV/ZIKV-induced CD4 + and CD8 + T-cell responses contribute to the observed protection but may not be essential for protection during ZIKV challenge. However, vaccination of Rag1 KO, Tcra KO, and muMt - mice demonstrated the critical role for ARPV/ZIKV-induced T-cells in developing protective immune responses following vaccination. Overall, both humoral and T-cell-mediated responses induced by ISFV-based vaccines are important for comprehensive immunity, and ISFV platforms continue to be a promising method for future vaccine development.

Rational Development of Live-Attenuated Zika Virus Vaccines.

Zika virus (ZIKV), a re-emerging mosquito-borne flavivirus, has caused outbreaks in Africa, Asia, the Pacific, and, more recently, in the Americas. ZIKV has been associated with the neurological autoimmune disorder Guillain-Barre syndrome in adults and congenital Zika syndrome in fetuses and infants, including microcephaly, spontaneous abortion, and intrauterine growth restriction. It is considered to be a major threat to global public health due to its unprecedented clinical impact on humans. Currently, there are no specific prophylactics or therapeutics available to prevent or treat ZIKV infection. The development of a safe and efficacious ZIKV vaccine remains a global health priority. Since the recent outbreak, multiple platforms have been used in the development of candidate ZIKV vaccines. The candidate vaccines have been shown to elicit strong T cell and neutralization antibody responses and protect against ZIKV infection in animal models. Some candidates have progressed successfully to clinical trials. Live-attenuated vaccines, which induce rapid and durable protective immunity, are one of the most important strategies for controlling flavivirus diseases. In this review, we discuss recent progress in the development of candidate live-attenuated ZIKV vaccines.

Post-Cure Polymerization and Depth-of-Cure Behaviors of Dental Bulk-Fill Resin-Based Composites.

INTRODUCTION: Polymerization for modern dental resin-based composites (RBCs) not only occurs immediately upon light exposure but also continues for another 24 hours, well beyond after light is terminated. However, many questions still remain about the role of polymerization kinetics in optimizing the physical properties of a new RBC type-the bulk-fill. OBJECTIVE: The aim is to study the post-cure polymerization kinetics of bulk-fill RBCs and to compare their degree of polymeric conversion (DC) and depth-of-cure (DoC) with an incremental-fill, conventional RBC. METHODS: Five representative bulk-fill RBCs [Surefil SDR+Stress Decreasing Resin Flow Plus (SDRFP), Tetric EvoCeram Bulk Fill (TECB), Filtek 1 Bulk Fill (F1B), Venus Bulk Fill (VB), and Sonicfill (SF3)] and one conventional RBC [Filtek Supreme Ultra (FSU)] were investigated. The upper surface per RBC specimen was exposed to a light curing unit (Paradigm, 3M-ESPE, irradiance=1221 ± 5 mW/cm2) for 20 seconds. The DC per RBC brand were measured at the bottom surface (specimen Ø=4 mm, thickness=3 mm and 5 mm) as a function of post-curing times using a Fourier transform infrared attenuated total reflection spectrometer. Real-time data recording for post-cure DC began immediately upon light exposure and continued at steady intervals, up to15 min, then again after 24 hours. The DoC of all six RBC brands (n=6 / group) were measured according to ISO-4049. Data were analyzed with nonlinear regression and analysis of variances (ANOVA)/Tukey (α=0.05). RESULTS: Mean DC for the six RBCs with 5 mm curing height after 24 hours were: TECB=79.5%, VB=75.7%, SDRFP=69.2%, SF3=65.8%, F1B=51.8%, and FSU=44.0%. Bulk-fill RBCs showed higher DC efficiency than the conventional RBC for both the 3 mm and 5 mm curing heights. Significant differences in DoC were found amongst the six RBC brands: VB=5.1 mm, SDRFP=4.6 mm, F1B=3.8 mm, TECB=3.5 mm, FSU=3.0 mm, and SF3=2.7 mm. CONCLUSION: DCs were more affected by specimen thickness, through which the curing light was attenuated, than RBC types. Clinician should be aware not all bulk-fill RBCs have a DoC greater than or equal to 4 mm. Also, a bulk-fill RBC that has a high DC after a post-cure time of 24 hours may not have a high DoC, which is typically measured relatively soon after light exposure.

The role of calcium-sensing receptor signaling in regulating transepithelial calcium transport.

The calcium-sensing receptor (CaSR) plays a critical role in sensing extracellular calcium (Ca2+) and signaling to maintain Ca2+ homeostasis. In the parathyroid, the CaSR regulates secretion of parathyroid hormone, which functions to increase extracellular Ca2+ levels. The CaSR is also located in other organs imperative to Ca2+ homeostasis including the kidney and intestine, where it modulates Ca2+ reabsorption and absorption, respectively. In this review, we describe CaSR expression and its function in transepithelial Ca2+ transport in the kidney and intestine. Activation of the CaSR leads to G protein dependent and independent signaling cascades. The known CaSR signal transduction pathways involved in modulating paracellular and transcellular epithelial Ca2+ transport are discussed. Mutations in the CaSR cause a range of diseases that manifest in altered serum Ca2+ levels. Gain-of-function mutations in the CaSR result in autosomal dominant hypocalcemia type 1, while loss-of-function mutations cause familial hypocalciuric hypercalcemia. Additionally, the putative serine protease, FAM111A, is discussed as a potential regulator of the CaSR because mutations in FAM111A cause Kenny Caffey syndrome type 2, gracile bone dysplasia, and osteocraniostenosis, diseases that are characterized by hypocalcemia, hypoparathyroidism, and bony abnormalities, i.e. share phenotypic features of autosomal dominant hypocalcemia. Recent work has helped to elucidate the effect of CaSR signaling cascades on downstream proteins involved in Ca2+ transport across renal and intestinal epithelia; however, much remains to be discovered.

Renal claudin-14 expression is not required for regulating Mg2+ balance in mice.

The kidneys play a crucial role in maintaining Ca2+ and Mg2+ homeostasis by regulating these minerals' reabsorption. In the thick ascending limb of Henle's loop (TAL), Ca2+ and Mg2+ are reabsorbed through the tight junctions by a shared paracellular pathway formed by claudin-16 and claudin-19. Hypercalcemia activates the Ca2+-sensing receptor (CaSR) in the TAL, causing upregulation of pore-blocking claudin-14 (CLDN14), which reduces Ca2+ and Mg2+ reabsorption from this segment. In addition, a high-Mg2+ diet is known to increase both urinary Mg2+ and Ca2+ excretion. Since Mg2+ may also activate CaSR, we aimed to investigate whether CaSR-dependent increases in CLDN14 expression also regulate urinary Mg2+ excretion in response to hypermagnesemia. Here, we show that a Mg2+-enriched diet increased urinary Mg2+ and Ca2+ excretion in mice; however, this occurred without detectable changes in renal CLDN14 expression. The administration of a high-Mg2+ diet to Cldn14-/- mice did not cause more pronounced hypermagnesemia or significantly alter urinary Mg2+ excretion. Finally, in vitro evaluation of CaSR-driven Cldn14 promoter activity in response to increasing Mg2+ concentrations revealed that Cldn14 expression only increases at supraphysiological extracellular Mg2+ levels. Together, these results suggest that CLDN14 is not involved in regulating extracellular Mg2+ balance following high dietary Mg2+ intake.NEW & NOTEWORTHY Using transgenic models and in vitro assays, this study examined the effect of Mg2+ on regulating urinary excretion of Ca2+ and Mg2+ via activation of the Ca2+-sensing receptor-claudin 14 (CLDN14) pathway. The study suggests that CLDN14 is unlikely to play a significant role in the compensatory response to hypermagnesemia.

Raynaud's phenomenon of the nipple as a side-effect of labetalol: Case report and literature review.

Raynaud's phenomenon of the nipple is an unusual side-effect of labetalol use. There is one official report from the United Kingdom. The present case concerns a 30-year-old woman, gravida 1, para 0, who developed pre-eclampsia and was treated with labetalol but subsequently reported neuropathic pain of the nipple. Nifedipine was then started as part of her treatment plan for blood pressure control and she no longer reported pain, despite being given six more doses of labetalol. Nifedipine is the first-line of treatment for Raynaud's phenomenon. The concomitant use of labetalol with nifedipine by a woman with Raynaud's phenomenon of the nipple has not been discussed before. The goal of this review is to raise awareness of this drug-induced phenomenon and to add to the limited literature available on this subject.

Does the future of laparoscopic sleeve gastrectomy lie in the outpatient surgery center? A retrospective study of the safety of 3162 outpatient sleeve gastrectomies.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) is a safe and effective procedure that can be performed as an outpatient procedure. OBJECTIVES: The aim of the study was to determine whether same-day discharge LSG is safe when performed in an outpatient surgery center. SETTING: Outpatient surgery centers. METHODS: The medical records of 3162 patients who underwent primary LSG procedure by 21 surgeons at 9 outpatient surgery centers from January 2010 through February 2018 were retrospectively reviewed. RESULTS: Three thousand one hundred sixty-two patients were managed with enhanced recovery after surgery protocol and were included in this analysis. The mean age and preoperative body mass index were 43.1 ± 10.8 years and 42.1 ± 7.1 kg/m2, respectively. Sleep apnea, type 2 diabetes, gastroesophageal reflux disease, hypertension, and hyperlipidemia were seen in 14.4%, 13.5%, 24.7%, 30.4%, and 17.6% patients, respectively. The mean total operative time was 56.4 ± 16.9 minutes (skin to skin). One intraoperative complication (.03%) occurred. The hospital transfer rate was .2%. The 30-day follow-up rate was 85%. The postoperative outcomes were analyzed based on the available data. The 30-day readmission, reoperation, reintervention, and emergency room visit rates were .6%, .6%, .2%, and .1%, respectively. The 30-day mortality rate was 0%. The total short-term complication rate was 2.5%. CONCLUSIONS: Same-day discharge seems to be safe when performed in an outpatient surgery center in selected patients. It would appear that outpatient surgery centers are a viable option for patients with minimal surgical risks.

Abdominal pregnancy in a low-resource setting.

BACKGROUND: Abdominal pregnancies are rare types of ectopic pregnancies with high rates of maternal mortality and morbidity when encountered anywhere in the world. Combining such high-risk pregnancy with the limitations found in a low-resource setting can lead to increased complications, which can be more difficult to manage. CASE: A 21-year-old woman, gravida 2 para 1001, at 36 weeks of gestation presented with a complicated abdominal pregnancy in Cameroon. The patient was followed through multiple complicated surgeries and medical treatments, including chemotherapy, until final resolution and complete recovery. CONCLUSION: This case highlights known complications of advanced abdominal pregnancy, including blood loss and infection. It also emphasizes limitations within the low-resource setting, which can independently add to the patient's morbidity and complicate management, and demonstrates examples of approaches that can be used aid management decisions within low-resource areas.

A randomized controlled trial of early versus delayed skin staple removal following caesarean section in the obese patient.

OBJECTIVE: To compare wound complications after Caesarean section in the obese patient, following early versus delayed skin staple removal. METHODS: We conducted a single-centre, non-inferiority, randomized controlled trial. Following Caesarean section, obese women (BMI ≥ 30 kg/m(2)) with subcutaneous wound depth ≥ 2.0 cm and skin staple closure of a transverse incision were randomized to staple removal on postoperative day 3 (early) or between postoperative day 7 and postoperative day 10 (delayed). The primary outcome was superficial wound dehiscence; a rate of 8% or higher in the early group was defined as inferior. Secondary outcomes were seroma/hematoma, surgical site infection, and visual analogue pain score. The planned sample size was 250 patients per group. RESULTS: The study was halted after 295 patients were randomized because of slow enrolment and exhaustion of funding. The rate of superficial wound dehiscence was 15.2% in the early group (n = 145) versus 11.5% in the delayed group (n = 148). The point estimate for this difference (3.7 %; 95% CI -4.4 to 12.4) favours delayed removal. However, because the 95% CI includes zero and the upper CI exceeds the predefined limit for non-inferiority (8%), non-inferiority was not demonstrated. The rates of all secondary outcomes were similar in the early group and the delayed group: seroma/hematoma (6.9% vs. 4.7%; RR 1.4, 95% CI 0.6 to 3.7, P = 0.4); surgical site infection (9.7% vs. 4.8%; RR 2.0, 95% CI 0.8 to 4.9, P = 0.1); and composite (superficial wound dehiscence, seroma/hematoma, and surgical site infection) wound complication (17.2% vs. 12.8%; RR 1.3, 95% CI 0.8 to 2.3, P = 0.3). CONCLUSION: The non-inferiority of early skin staple removal was not demonstrated. Delayed removal of staples should remain the accepted standard in the obese patient following Caesarean section.

Objectif : Comparer les effets du retrait précoce des agrafes cutanées aux effets de leur retrait différé, en ce qui concerne les complications de plaie à la suite d'une césarienne chez une patiente obèse. Méthodes : Nous avons mené un essai comparatif randomisé de non-infériorité au sein d'un seul centre. À la suite d'une césarienne, les femmes obèses (IMC ≥ 30 kg/m2) qui présentaient une profondeur de plaie sous-cutanée ≥ 2,0 cm et une incision transversale fermée au moyen d'agrafes cutanées ont été affectées, au hasard, à un groupe devant se faire retirer ces agrafes trois jours à la suite de l'opération (retrait précoce) ou à un groupe devant se faire retirer ces agrafes entre le septième jour et le dixième jour suivant l'opération (retrait différé). La déhiscence superficielle de la plaie constituait le critère d'évaluation principal; un taux de 8 % ou plus au sein du groupe « retrait précoce ¼ a été défini comme étant inférieur. Les critères d'évaluation secondaires étaient le sérome / hématome, l'infection du champ opératoire et le score sur l'échelle visuelle analogue. La taille planifiée de l'échantillon était de 250 patientes par groupe. Résultats : L'étude a été suspendue à la suite de la randomisation de 295 patientes, en raison de la lenteur du processus de sollicitation des patientes et de l'épuisement des fonds. Le taux de déhiscence superficielle de la plaie était de 15,2 % au sein du groupe « retrait précoce ¼ (n = 145) vs 11,5 % au sein du groupe « retrait différé ¼ (n = 148). L'estimation ponctuelle pour cette différence (3,7 %; IC à 95 %, −4,4 - 12,4) favorise le retrait différé. Toutefois, puisque l'IC à 95 % contient zéro et que l'IC supérieur dépasse la limite prédéfinie pour ce qui est de la non-infériorité (8 %), cette dernière n'a pas été démontrée. Les taux de tous les critères d'évaluation secondaires étaient semblables dans les deux groupes : sérome / hématome (6,9 % vs 4,7 %; RR, 1,4, IC à 95 %, 0,6 - 3,7, P = 0,4); infection du champ opératoire (9,7 % vs 4,8 %; RR, 2,0, IC à 95 %, 0,8 - 4,9, P = 0,1); et complication composite de la plaie (déhiscence superficielle de la plaie, sérome / hématome, infection du champ opératoire) (17,2 % vs 12,8 %; RR, 1,3, IC à 95 %, 0,8 - 2,3, P = 0,3). Conclusion : La non-infériorité du retrait précoce des agrafes cutanées n'a pas été démontrée. Le retrait différé des agrafes devrait demeurer la norme acceptée en ce qui concerne les patientes obèses à la suite d'une césarienne.

Quality of life of older African American women in rural North Carolina.

PURPOSE: The purpose of this study was to investigate the relationship of activity to the quality of life of female African American elders. METHODS: Framed in the critical emancipatory paradigm, the study used qualitative research methodology, including the analysis of transcribed interviews and the critical review of relevant literature. RESULTS: Contributors to this study identified numerous past and present activities. The activities were bound together with the themes of "The Lord," Doing, Family, Environment, "I had to let it go," and "Just a part of life." DISCUSSION: Study results indicate that participants have enhanced their quality of life and emancipated themselves through engagement in a variety of activities that hold both real and symbolic meaning.