Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity.

The papain-like protease PLpro is an essential coronavirus enzyme that is required for processing viral polyproteins to generate a functional replicase complex and enable viral spread1,2. PLpro is also implicated in cleaving proteinaceous post-translational modifications on host proteins as an evasion mechanism against host antiviral immune responses3-5. Here we perform biochemical, structural and functional characterization of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro (SCoV2-PLpro) and outline differences with SARS-CoV PLpro (SCoV-PLpro) in regulation of host interferon and NF-κB pathways. SCoV2-PLpro and SCoV-PLpro share 83% sequence identity but exhibit different host substrate preferences; SCoV2-PLpro preferentially cleaves the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. The crystal structure of SCoV2-PLpro in complex with ISG15 reveals distinctive interactions with the amino-terminal ubiquitin-like domain of ISG15, highlighting the high affinity and specificity of these interactions. Furthermore, upon infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses. Notably, inhibition of SCoV2-PLpro with GRL-0617 impairs the virus-induced cytopathogenic effect, maintains the antiviral interferon pathway and reduces viral replication in infected cells. These results highlight a potential dual therapeutic strategy in which targeting of SCoV2-PLpro can suppress SARS-CoV-2 infection and promote antiviral immunity.

The interferon-regulated host factor hnRNPA0 modulates HIV-1 production by interference with LTR activity, mRNA trafficking, and programmed ribosomal frameshifting.

The interplay between host factors and viral components impacts viral replication efficiency profoundly. Members of the cellular heterogeneous nuclear ribonucleoprotein family (hnRNPs) have been extensively studied as HIV-1 host dependency factors, but whether they play a role in innate immunity is currently unknown. This study aimed to identify hnRNPA0 as a type I interferon (IFN)-repressed host factor in HIV-1-infected cells. Knockdown of hnRNPA0, a situation that mirrors conditions under IFN stimulation, increased LTR activity, export of unspliced HIV-1 mRNA, viral particle production, and thus, increased infectivity. Conversely, hnRNPA0 overexpression primarily reduced plasmid-driven and integrated HIV-1 long terminal repeat (LTR) activity, significantly decreasing total viral mRNA and protein levels. In addition, high levels of hnRNPA0 significantly reduced the HIV-1 programmed ribosomal frameshifting efficiency, resulting in a shift in the HIV-1 p55/p15 ratio. The HIV-1 alternative splice site usage remained largely unaffected by altered hnRNPA0 levels suggesting that the synergistic inhibition of the LTR activity and viral mRNA transcription, as well as impaired ribosomal frameshifting efficiency, are critical factors for efficient HIV-1 replication regulated by hnRNPA0. The pleiotropic dose-dependent effects under high or low hnRNPA0 levels were further confirmed in HIV-1-infected Jurkat cells. Finally, our study revealed that hnRNPA0 levels in PBMCs were lower in therapy-naive HIV-1-infected individuals compared to healthy controls. Our findings highlight a significant role for hnRNPA0 in HIV-1 replication and suggest that its IFN-I-regulated expression levels are critical for viral fitness allowing replication in an antiviral environment.IMPORTANCERNA-binding proteins, in particular, heterogeneous nuclear ribonucleoproteins (hnRNPs), have been extensively studied. Some act as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. Our study revealed hnRNPA0 as an IFN-regulated host factor, that is differently expressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naïve HIV-1-infected individuals. Our findings demonstrate the significant pleiotropic role of hnRNPA0 in viral replication: In high concentrations, hnRNPA0 limits viral replication by negatively regulating Tat-LTR transcription, retaining unspliced mRNA in the nucleus, and significantly impairing programmed ribosomal frameshifting. Low hnRNPA0 levels as observed in IFN-treated THP-1 cells, particularly facilitate HIV LTR activity and unspliced mRNA export, suggesting a role in innate immunity in favor of HIV replication. Understanding the mode of action between hnRNPA0 and HIV-1 gene expression might help to identify novel therapeutically strategies against HIV-1 and other viruses.

Enhanced recovery after surgery (ERAS) in colorectal surgery: implementation is still beneficial despite modern surgical and anesthetic care.

PURPOSE: Enhanced recovery after surgery (ERAS) protocols have shown beneficial outcomes in the last 20 years. Nevertheless, simultaneously implemented technical improvements such as minimally invasive access or modified anesthesia care may play a crucial role in optimizing patient outcome. The aim of the study was to investigate the effect of ERAS implementation in a highly specialized colorectal center. METHODS: This is a propensity score matched single-center study comparing the short-term outcomes of patients undergoing elective colorectal surgery in a society-indepedent ERAS program from January 2021 to August 2022 to standard perioperative care from January 2019 to December 2020. RESULTS: Four hundred fifty-six patients were included in the propensity score matched analysis with 228 patients per group (ERAS vs. standard care). Minimally invasive access was used in 80.2% vs. 77.6% (p = 0.88), and there were 16.6% vs. 18.8% (p = 0.92) rectal procedures in the ERAS and standard care group, respectively. Major complications occurred in 10.1% vs. 11.4% (p = 0.65) and anastomotic leakage demanding operative revision in 2.2% vs. 2.6% (p = 0.68) in the ERAS and standard care group, respectively. ERAS lead to a lower number of non-surgical complications compared to standard care (57 vs. 79; p = 0.02). Mean length of stay (LOS) and mean costs per case were lower in ERAS compared to standard care (9.2 ± 5.6 days vs. 12.7 ± 7.4 days, p < 0.01; costs 33,727 ± 15,883 USD vs. 40,309 ± 29,738 USD, p < 0.01). CONCLUSION: The implementation of an ERAS protocol may lead to a reduction of LOS, costs, and a lower number of non-surgical complications even in a highly specialized colorectal unit using modern surgical and anesthetic care. ( ClinialTrials.gov number NCT05773248).

Low serum iPTH at the end of surgery is the earliest predictor of postoperative hypocalcemia after total thyroidectomy.

BACKGROUND: The most frequent complication of total thyroidectomy remains hypocalcemia due to low postoperative levels of serum intact parathyroid hormone (iPTH). The purpose of this study was to investigate the role of decreased iPTH at the end of surgery in predicting hypocalcemia. In addition, we examined the percentage decrease of iPTH as potential indicator of hypocalcemia. METHODS: We retrospectively collected the data of patients who underwent total thyroidectomy for benign and malignant diseases at our institution between 2010 and 2022. The iPTH level was measured before and at the end of surgery, and serum calcium levels on the first postoperative day. Demographic, clinical, and biochemical characteristics of patients with low iPTH were compared with patients with normal iPTH levels using ANOVA for continuous variables and χ2-tests for categorical variables. Multivariable logistic regression analysis evaluated the association of iPTH at the end of surgery and the relative reduction of iPTH with postoperative hypocalcemia. RESULTS: The mean age of the 607 patients in this study was 55.6 years, and the female-to-male ratio was 5:1. Goiter was the most common indication for surgery (N = 382, 62.9%), followed by Graves' disease (N = 135, 22.2%). The mean preoperative iPTH was 49.0 pg/ml, while the mean postoperative iPTH was 29.3 pg/ml. A total of 197 patients (32.5%) had an iPTH level below normal, 77 patients (39%), had iPTH levels of 10-15.0 pg/ml and 120 patients (61%) of < 10.0 pg/ml at the end of surgery. Among all patients, 124 (20.4%) developed hypocalcemia on the first postoperative day. The mean percentage of decrease of iPTH was highest among patients with iPTH < 10 pg/ml (76.9%, p < 0.01); this group of patients had also the highest rate of postoperative hypocalcemia on day one (45.0% vs. 26.0% vs 12.2%, p < 0.01). CONCLUSIONS: Measurement of iPTH at the end of total thyroidectomy predicts patients who are at risk for postoperative hypocalcemia. The combination of low serum iPTH with a decrease in iPTH level of ≥ 50% may improve prediction of hypocalcemia compared to iPTH levels alone allowing for early calcium substitution in these patients at high risk of developing postoperative hypocalcemia.

Routine use of intraoperative nerve monitoring is associated with a reduced risk of vocal cord dysfunction after thyroid cancer surgery.

BACKGROUND: The aim of this study was to investigate the associations between individual surgeon's intraoperative nerve monitoring (IONM) practice and factors associated with vocal cord (VC) dysfunction in patients with thyroid cancer undergoing thyroidectomy. METHODS: Using Collaborative Endocrine Surgery Quality Improvement Program (CESQIP) 2014-21 data, multivariable logistic regression analyses investigated variables associated with short- and long-term VC-dysfunction, associations of routine use of IONM with postoperative outcomes, and patient characteristics associated with IONM use. RESULTS: Among 5,446 patients (76.7% female, mean age 49 years), 68.5% had surgery by surgeons using IONM in ≥ 90% of cases (63% of surgeons, n = 73). Post-operative VC-dysfunction was diagnosed by laryngoscopy in 3.0% of patients in the short-term and 2.7% in the long-term. When surgeons routinely used IONM, the incidence of VC-dysfunction was 2.4% in the short-term and 2.2% in the long-term, compared to 4.4% and 3.7%, respectively, when surgeons did not routinely use IONM (p < 0.01). After adjustment, routine use of IONM was independently associated with reduced risk of short- (OR 0.48, p < 0.01) and long-term (OR 0.52, p < 0.01) VC-dysfunction, a lower risk of postoperative hypoparathyroidism in the short- (OR 0.67, p < 0.01) and long-term (OR 0.54, p < 0.01), and higher likelihood of same-day discharge (OR 2.03, p < 0.01). Extrathyroidal tumor extension and N1-stage were factors associated with postoperative VC-dysfunction in the short- (OR 3.12, p < 0.01; OR 1.92, p = 0.01, respectively) and long-term (OR 3.11, p < 0.01; OR 2.32, p < 0.01, respectively). CONCLUSION: Routine use of IONM was independently associated with a lower risk of endocrine surgery-specific complications and greater likelihood of same-day discharge.

The prognostic significance of CXCR4 and SDF-1 in differentiated thyroid cancer depends on CD8+ density.

BACKGROUND: Tumor infiltration with cytotoxic CD8+ T-cells is associated with a favorable outcome in several neoplasms, including thyroid cancer. The chemokine axis CXCR4/SDF-1 correlates with more aggressive tumors, but little is known concerning the prognostic relevance in relation to the tumor immune microenvironment of differentiated thyroid cancer (DTC). METHODS: A tissue microarray (TMA) of 37 tumor specimens of primary DTC was analyzed by immunohistochemistry (IHC) for the expression of CD8+, CXCR4, phosphorylated CXCR4 and SDF-1. A survival analysis was performed on a larger collective (n = 456) at RNA level using data from The Cancer Genome Atlas (TCGA) papillary thyroid cancer cohort. RESULTS: Among the 37 patients in the TMA-cohort, the density of CD8+ was higher in patients with less advanced primary tumors (median cells/TMA-punch: 12.5 (IQR: 6.5, 12.5) in T1-2 tumors vs. 5 (IQR: 3, 8) in T3-4 tumors, p = 0.05). In the TCGA-cohort, CXCR4 expression was higher in patients with cervical lymph node metastasis compared to N0 or Nx stage (CXCR4high/low 116/78 vs. 97/116 vs. 14/35, respectively, p = 0.001). Spearman's correlation analysis of the TMA-cohort demonstrated that SDF-1 was significantly correlated with CXCR4 (r = 0.4, p = 0.01) and pCXCR4 (r = 0.5, p = 0.002). In the TCGA-cohort, density of CD8+ correlated with CXCR4 and SDF-1 expression (r = 0.58, p < 0.001; r = 0.4, p < 0.001). The combined marker analysis of the TCGA cohort demonstrated that high expression of both, CXCR4 and SDF-1 was associated with reduced overall survival in the CD8 negative TCGA cohort (p = 0.004). CONCLUSION: These findings suggest that the prognostic significance of CXCR4 and SDF-1 in differentiated thyroid cancer depends on the density of CD8 positive T-lymphocytes. Further studies with larger sample sizes are needed to support our findings and inform future investigations of new treatment and diagnostic options for a more personalized approach for patients with differentiated thyroid cancer.

Robot-assisted vs. laparoscopic repair of complete upside-down stomach hiatal hernia (the RATHER-study): a prospective comparative single center study.

BACKGROUND: Complete upside-down stomach (cUDS) hernias are a subgroup of large hiatal hernias characterized by high risk of life-threatening complications and technically challenging surgical repair including complex mediastinal dissection. In a prospective, comparative clinical study, we evaluated intra- and postoperative outcomes, quality of life and symptomatic recurrence rates in patients with cUDS undergoing robot-assisted, as compared to standard laparoscopic repair (the RATHER-study). METHODS: All patients with cUDS herniation requiring elective surgery in our institution between July 2015 and June 2019 were evaluated. Patients undergoing primary open surgery or additional associated procedures were not considered. Primary endpoints were intra- and postoperative complications, 30-day morbidity, and mortality. During the 8-53 months follow-up period, patients were contacted by telephone to assess symptoms associated to recurrence, whereas quality of life was evaluated utilizing the Gastroesophageal Reflux Disease-Health-Related Quality of Life (GERD-HRQL) questionnaire. RESULTS: A total of 55 patients were included. 36 operations were performed with robot-assisted (Rob-G), and 19 with standard laparoscopic (Lap-G) technique. Patients characteristics were similar in both groups. Median operation time was 232 min. (IQR: 145-420) in robot-assisted vs. 163 min. (IQR:112-280) in laparoscopic surgery (p < 0.001). Intraoperative complications occurred in 5/36 (12.5%) cases in the Rob-G group and in 5/19 (26%) cases in the Lap-G group (p = 0.28). No conversion was necessary in either group. Minor postoperative complications occurred in 13/36 (36%) Rob-G patients and 4/19 (21%) Lap-G patients (p = 0.36). Mortality or major complications did not occur in either group. Two asymptomatic recurrences were observed in the Rob-G group only. No patient required revision surgery. Finally, all patients expressed satisfaction for treatment outcome, as indicated by similar GERD-HRQL scores. CONCLUSION: While robot-assisted surgery provides additional precision, enhanced visualization, and greater feasibility in cUDS hiatal hernia repair, its clinical outcome is at least equal to that obtained by standard laparoscopic surgery.

From open Ivor Lewis esophagectomy to a hybrid robotic-assisted thoracoscopic approach: a single-center experience over two decades.

PURPOSE: Robotic-assisted procedures are increasingly used in esophageal cancer surgery. We compared postoperative complications and early oncological outcomes following hybrid robotic-assisted thoracoscopic esophagectomy (Rob-E) and open Ivor Lewis esophagectomy (Open-E), performed in a single mid-volume center, in the context of evolving preoperative patient and tumor characteristics over two decades. METHODS: We evaluated prospectively collected data from a single center from 1999 to 2020 including 321 patients that underwent Ivor Lewis esophagectomy, 76 underwent Rob-E, and 245 Open-E. To compare perioperative outcomes, a 1:1 case-matched analysis was performed. Endpoints included postoperative morbidity and 30-day mortality. RESULTS: Preoperative characteristics revealed increased rates of adenocarcinomas and wider use of neoadjuvant treatment over time. A larger number of patients with higher ASA grades were operated with Rob-E. In case-matched cohorts, there were no differences in the overall morbidity (69.7% in Rob-E, 60.5% in Open-E, p value 0.307), highest Clavien-Dindo grade per patient (43.4% vs. 38.2% grade I or II, p value 0.321), comprehensive complication index (median 20.9 in both groups, p value 0.401), and 30-day mortality (2.6% in Rob-E, 3.9% in Open-E, p value 1.000). Similar median numbers of lymph nodes were harvested (24.5 in Rob-E, 23 in Open-E, p value 0.204), and comparable rates of R0-status (96.1% vs. 93.4%, p value 0.463) and distribution of postoperative UICC stages (overall p value 0.616) were observed. CONCLUSIONS: Our study demonstrates similar postoperative complications and early oncological outcomes after Rob-E and Open-E. However, the selection criteria for Rob-E appeared to be less restrictive than those of Open-E surgery.

Limited Neutralization of Authentic Severe Acute Respiratory Syndrome Coronavirus 2 Variants Carrying E484K In Vitro.

Whether monoclonal antibodies are able to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has been investigated using pseudoviruses. In this study we show that bamlanivimab, casirivimab, and imdevimab efficiently neutralize authentic SARS-CoV-2, including variant B.1.1.7 (alpha), but variants B.1.351 (beta) and P.2 (zeta) were resistant against bamlanivimab and partially resistant to casirivimab. Whether antibodies are able to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variantshas been investigated using pseudoviruses. We show that authentic SARS-CoV-2 carrying E484K were resistant against bamlanivimab and less susceptible to casirivimab, convalescent and vaccine-elicited sera.

Localization of Drosophila CENP-A to non-centromeric sites depends on the NuRD complex.

Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

Scenarios for the Spread of COVID-19 in Manaus, Northern Brazil.

We use a compartmental model to project scenarios for the spread of COVID-19 in the city of Manaus, with different degrees of social distancing and isolation of infectious individuals. In a scenario without any containment measures, our projections point to more than 50,000 (simultaneously) infectious individuals before the end of April 2020. Only in a scenario with strict and extensive social distancing (lockdown), implemented and observed as of March 23rd, 2020, would this number be less than 50,000 in the first half of June. Furthermore, an increase in the number of tests or in the collective awareness among the population regarding the importance of strict distancing measures would have only had a significant impact if implemented before the end of March.

Duodenal localization is a negative predictor of survival after small bowel adenocarcinoma resection: A population-based, propensity score-matched analysis.

BACKGROUND AND OBJECTIVES: This study assessed the influence of tumor localization of small bowel adenocarcinoma on survival after surgical resection. METHODS: Patients with resected small bowel adenocarcinoma, ACJJ stage I-III, were identified from the Surveillance, Epidemiology, and End Results database from 2004 to 2013. The impact of tumor localization on overall and cancer-specific survival was assessed using Cox proportional hazard regression models with and without risk-adjustment and propensity score methods. RESULTS: Adenocarcinoma was localized to the duodenum in 549 of 1025 patients (53.6%). There was no time trend for duodenal localization (P = 0.514). The 5-year cancer-specific survival rate was 48.2% (95%CI: 43.3-53.7%) for patients with duodenal carcinoma and 66.6% (95%CI: 61.6-72.1%) for patients with cancer located in the jejunum or ileum. Duodenal localization was associated with worse overall and cancer-specific survival in univariable (HR = 1.73; HR = 1.81, respectively; both P < 0.001), multivariable (HR = 1.52; HR = 1.65; both P < 0.001), and propensity score-adjusted analyses (HR = 1.33, P = 0.012; HR = 1.50, P = 0.002). Furthermore, young age, retrieval of more than 12 regional lymph nodes, less advanced stage, and married matrimonial status were positive, independent prognostic factors. CONCLUSIONS: Duodenal localization is an independent risk factor for poor survival after resection of adenocarcinoma.

Nerve monitoring in endocrine surgery: Practice patterns differ among surgeons for parathyroidectomy and thyroidectomy.

BACKGROUND: It is unknown whether intraoperative nerve monitoring is associated with reduced vocal cord dysfunction after parathyroidectomy. We aimed to investigate intraoperative nerve monitoring use among Collaborative Endocrine Surgery Quality Improvement Program surgeons and factors associated with vocal cord dysfunction after parathyroidectomy. METHODS: Patients who underwent parathyroidectomy included in the Collaborative Endocrine Surgery Quality Improvement Program (2014-2022) were identified. The annual percent change in parathyroidectomies performed with intraoperative nerve monitoring was calculated using joinpoint regression. Multivariable logistic regression was used to compare outcomes between patients undergoing parathyroidectomy with/without intraoperative nerve monitoring. To compare surgeon-specific trends, Collaborative Endocrine Surgery Quality Improvement Program thyroidectomy and parathyroidectomy datasets (2014-2021) were combined. Parathyroidectomies performed by surgeons who used intraoperative nerve monitoring consistently in thyroidectomy were identified. Factors associated with intraoperative nerve monitoring were examined using multivariable logistic regression. RESULTS: A total of 9,813 patients underwent parathyroidectomy. Intraoperative nerve monitoring was used in 49% of cases (n = 4,818). There was an increase in parathyroidectomies with intraoperative nerve monitoring from 2014 to 2018 (annual percent change 22.2, P = .01), followed by a plateau (2018-2022 annual percent change -0.66, P = .85). Few patients (0.44%, n = 43) developed vocal cord dysfunction. Vocal cord dysfunction was not associated with intraoperative nerve monitoring (adjusted odds ratio 0.92, P = .75). Whereas 41% (n = 56/138) of surgeons used intraoperative nerve monitoring routinely in parathyroidectomy, 65% (n = 90/138) used it routinely in thyroidectomy. Among surgeons who used intraoperative nerve monitoring routinely in thyroidectomy, only 57% used it routinely in parathyroidectomy; factors associated with intraoperative nerve monitoring during parathyroidectomy included reoperation (adjusted odds ratio 2.51, P < .01), secondary/tertiary hyperparathyroidism (adjusted odds ratio 1.42, P = .02), multiglandular disease (adjusted odds ratio 1.76, P < .001), and non-localized disease (adjusted odds ratio 1.65, P < .001). CONCLUSION: Endocrine surgeons use intraoperative nerve monitoring selectively. Surgeons who routinely use intraoperative nerve monitoring during thyroidectomy are more likely to use it during parathyroidectomy. Future studies should determine who may benefit most from intraoperative nerve monitoring in parathyroidectomy.

IPMN: surgical treatment.

PURPOSE: Cystic pancreatic tumors are being detected more frequently, and particularly, intraductal papillary mucinous neoplasia (IPMN) has recently attracted increased attention. The detection rate of IPMN has increased over the last decade; however, management of this neoplasm remains controversial. METHODS: Based on a review of the relevant literature and the international guidelines, we discuss the diagnostic evaluation of IPMN, its treatment, and prognosis. RESULTS: While IPMN represents only a distinct minority of all pancreatic cancers, they appear to be a relatively frequent neoplastic form of pancreatic cystic neoplasm. It may not be possible to differentiate main duct disease from branch duct disease (MD-IPMN vs. BD-IPMN) prior to surgery. This distinction has not only an impact on treatment but also on prognosis, as MD-IPMN is more often malignant. IPMN has updated consensus guideline indications for conservative and surgical resection. CONCLUSIONS: Since patients with IPMN of the pancreas are at risk of developing recurrent IPMN and pancreatic ductal adenocarcinoma in the remnant pancreas and extrapancreatic malignancies, early recognition, treatment, and systemic surveillance are of great importance. No conclusions can be drawn from the available evidence with respect to the efficacy of surveillance and follow-up treatment programs. A better understanding of the natural course of IPMN and the biology of pancreatic cancer is mandatory to enable further diagnostic and treatment improvements.

The VBNC state: a fundamental survival strategy of Acinetobacter baumannii.

IMPORTANCE: Currently, the viable but non-culturable (VBNC) state is an underappreciated niche for pathogenic bacteria which provides a continuous source for recurrent infections and transmission. We propose the VBNC state to be a global persistence mechanism used by various A. baumannii strains to cope with many stresses it is confronted with in the clinical environment and in the host. This requires a novel strategy to detect viable cells of this pathogen that is not only based on plating assays.

Using a stress reservoir to improve urine leakage after artificial urinary sphincter implantation.

INTRODUCTION: Although the longest efficacy record, some patients report about urinary leakage during higher intra-abdominal pressure after Artificial Urinary Sphincter (AUS) implantation. To improve the continent results, we placed in addition to the occluding cuff and the pressure regulating balloon, in a second procedure a stress-relief reservoir in lower abdomen, for additional passive pressure transmission to the occluding cuff. METHODS: In this retrospective monocentric data analysis between 2011 and 2018, 80 patients with persistence incontinence after AUS implantation were included. Stress-relief reservoir was indicated in 12 patients with involuntary leakage of urine, that occurred when intra-abdominal pressure raised. RESULTS: In all 12 cases, the stress reservoir was easily implanted and there were no intraoperative complications. In a mean follow-up time of 53 months, the pad per day usage (p/d) improved from 3 (± 1.2) to 1.7 (± 1.5) (p = 0.001). Two patients with multiple previous abdominal surgeries used an equal number of pads after SRR; however, an improvement during physical exertion was reported. Continence situation was evaluated with a questionnaire and was rated as "excellent", "good", or "satisfactory" by 11 (92%) patients. CONCLUSION: The persistence of urinary incontinence after AUS is a challenging topic. Implantation of a stress reservoir in carefully selected patients with urinary leakage during higher intra-abdominal pressure is minimally invasive and offers new options to improve the proven long-term record of AUS. Certainly, more investigations are needed to determine the clinical relevance of this approach.

Disease-Specific Survival Trends for Patients Presenting with Differentiated Thyroid Cancer and Distant Metastases in the United States, 1992-2018.

Objective: Differentiated thyroid cancer (DTC) is associated with an excellent prognosis, but patients with distant metastatic DTC have a 10-year disease-specific survival (DSS) of just 50%. The incidence of distant metastatic DTC has steadily increased in the United States since the 1980s. The aim of this study was to examine trends in survival and treatment for patients with distant metastatic DTC. Methods: In this population-based, retrospective cohort study, patients with distant metastatic DTC were identified from the Surveillance, Epidemiology, and End Results-13 cancer registry program. Multivariable logistic and Cox regression analyses were used to examine factors associated with DSS and management. Annual percentage changes in treatment patterns were calculated using log-linear regression. Results: During 1992-2018, 1991 patients (69.7% white, 58.0% female, 47.5% aged ≥65 years) were diagnosed with distant metastatic DTC. Papillary thyroid cancer was the most common histological type (74.5%). While the 10-year DSS for overall DTC increased over time (95.4% for patients diagnosed in 1992-1998, 96.6% in 1999-2008, and 97.3% in 2009-2018; p < 0.01), 10-year DSS for DTC with distant metastases did not change (50.2%, 47.3%, and 52.4%, respectively; p = 0.48). Ten-year DSS rates were reduced for patients aged ≥65 years (28.1%), patients undergoing nonsurgical treatment with external beam radiation therapy and/or systemic therapy (6.0%), and patients undergoing no/unknown treatment (32.8%). On multivariable analysis, oncocytic carcinoma, age 65-79 and ≥80 years, male sex, node-positive disease, larger tumor size, nonsurgical treatment, and no/unknown treatment were associated with increased risk of thyroid cancer death. Between 1992 and 2018, the rate of nonsurgical treatment increased, on average, 1.3% per year (1992-1998: 22.9% vs. 2009-2018: 25.6%; p = 0.03), and the rate of patients receiving no/unknown treatment increased 1.9% per year (1992-1998: 11.3% vs. 2009-2018: 15.6%; p = 0.01). Patients aged 65-79 and ≥80 years were more likely than younger patients to receive nonsurgical management or no/unknown treatment. Conclusion: Patients diagnosed with distant metastatic DTC have experienced no improvement in DSS over the past three decades. An increasing proportion of patients diagnosed with distant metastatic DTC are receiving nonsurgical treatment or no/unknown treatment over time; the proportion was highest among the oldest patients.

Comparison of the Ct-values for genomic and subgenomic SARS-CoV-2 RNA reveals limited predictive value for the presence of replication competent virus.

SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19. In addition to the full length positive-sensed, single-stranded genomic RNA (gRNA), viral subgenomic RNAs (sgRNAs) that are required for expression of the 3' region of the genome are synthesized in virus-infected cells. However, whether these sgRNA-species might be used as a measure of active virus replication and to predict infectivity is still under debate. The commonly used methods to monitor and quantitate SARS-CoV-2 infections are based on RT-qPCR analysis and the detection of gRNA. The infectivity of a sample obtained from nasopharyngeal or throat swabs is associated with the viral load and inversely correlates with Ct-values, however, a cut-off value predicting the infectivity highly depends on the performance of the assay. Furthermore, gRNA derived Ct-values result from nucleic acid detection and do not necessarily correspond to active replicating virus. We established a multiplex RT-qPCR assay on the cobas 6800 omni utility channel concomitantly detecting SARS-CoV-2 gRNAOrf1a/b, sgRNAE,7a,N, and human RNaseP-mRNA used as human input control. We compared the target specific Ct-values with the viral culture frequency and performed ROC curve analysis to determine the assay sensitivity and specificity. We found no advantage in the prediction of viral culture when using sgRNA detection compared to gRNA only, since Ct-values for gRNA and sgRNA were highly correlated and gRNA offered a slightly more reliable predictive value. Single Ct-values alone only provide a very limited prediction for the presence of replication competent virus. Hence, careful consideration of the medical history including symptom onset has to be considered for risk stratification.

Hypoxia-altered cholesterol homeostasis enhances the expression of interferon-stimulated genes upon SARS-CoV-2 infections in monocytes.

Hypoxia contributes to numerous pathophysiological conditions including inflammation-associated diseases. We characterized the impact of hypoxia on the immunometabolic cross-talk between cholesterol and interferon (IFN) responses. Specifically, hypoxia reduced cholesterol biosynthesis flux and provoked a compensatory activation of sterol regulatory element-binding protein 2 (SREBP2) in monocytes. Concomitantly, a broad range of interferon-stimulated genes (ISGs) increased under hypoxia in the absence of an inflammatory stimulus. While changes in cholesterol biosynthesis intermediates and SREBP2 activity did not contribute to hypoxic ISG induction, intracellular cholesterol distribution appeared critical to enhance hypoxic expression of chemokine ISGs. Importantly, hypoxia further boosted chemokine ISG expression in monocytes upon infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Mechanistically, hypoxia sensitized toll-like receptor 4 (TLR4) signaling to activation by SARS-CoV-2 spike protein, which emerged as a major signaling hub to enhance chemokine ISG induction following SARS-CoV-2 infection of hypoxic monocytes. These data depict a hypoxia-regulated immunometabolic mechanism with implications for the development of systemic inflammatory responses in severe cases of coronavirus disease-2019 (COVID-19).

High density of CXCL12-positive immune cell infiltration predicts chemosensitivity and recurrence-free survival in ovarian carcinoma.

BACKGROUND: Ovarian carcinoma is the most lethal gynecologic malignancy because of its late diagnosis, extremely high recurrence rate, and limited curative treatment options. In clinical practice, high-grade serous carcinoma (HGSC) predominates due to its frequency, high aggressiveness, and rapid development of drug resistance. Recent evidence suggests that CXCL12 is an important immunological factor in ovarian cancer progression. Therefore, we investigated the predictive and prognostic significance of the expression of this chemokine in tumor and immune cells in patients with HGSC. METHODS: We studied a cohort of 47 primary high-grade serous ovarian carcinomas and their associated recurrences. A tissue microarray was constructed to evaluate the CXCL12 immunostained tumor tissue. CXCL12 expression was evaluated and statistically analyzed to correlate clinicopathologic data, overall survival, and recurrence-free survival. RESULTS: A high proportion of CXCL12 + positive immune cells in primary ovarian serous carcinoma correlated significantly with chemosensitivity (p = 0.005), overall survival (p = 0.021), and longer recurrence-free survival (p = 0.038). In recurrent disease, high expression of CXCL12 was also correlated with better overall survival (p = 0.040). Univariate and multivariate analysis revealed that high CXCL12 + tumor-infiltrating immune cells (TICs) (HR 0.99, p = 0.042, HR 0.99, p = 0.023, respectively) and combined CXCL12 + /CD66b + infiltration (HR 0.15, p = 0.001, HR 0.13, p = 0.001, respectively) are independent favorable predictive markers for recurrence-free survival. CONCLUSION: A high density of CXCL12 + TICs predicts a good response to chemotherapy, leading to a better overall survival and a longer recurrence-free interval. Moreover, with concomitant high CXCL12/CD66b TIC density, it is an independent favorable predictor of recurrence-free survival in patients with ovarian carcinoma.