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BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5(+) cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Because of the COVID-19 pandemic, many transport kits have been manufactured to preserve and transport nasopharyngeal swab samples (NPSs) from patients. However, there is no information on the performance of the different virus transport media (VTM) used in COVID-19 diagnosis in the population of Santiago de Chile. We compared the RT-qPCR amplification profile of five different viral transport kit medium, including DNA/RNA Shield ™, NAT, VTM-N, Ezmedlab™, and Phosphate Buffered Saline (PBS), for nasopharyngeal swab samples (NPSs) from Central Metropolitan Health Service, Santiago, Chile. The DNA/RNA Shield™ medium showed a better performance in terms of Cq and RFU values for the internal reference RNase P and viral ORF1ab probes. By contrast, the PBS transport medium registered a higher Cq values for the viral and reference gene, compared to the other VTM. DNA/RNA Shield ™ shows higher relative fluorescence units (RFU) and lower Cq values for the reference gene. Collectively, our results suggest that PBS medium could compromise the sample diagnosis because its lower RT-qPCR performance. The NAT, Ezmedlab and VTM-N and DNA/RNA Shield ™ media show acceptable RT-qPCR parameters and, consequently, seem suitable for use in COVID-19 diagnosis. This article is protected by copyright. All rights reserved.

Chromatin structural organization, gene expression and proteostasis are intricately regulated in a wide range of biological processes, both physiological and pathological. Protein acetylation, a major post-translational modification, is tightly involved in interconnected biological networks, modulating the activation of gene transcription and protein action in cells. A very large number of studies describe the pivotal role of the so-called acetylome (accounting for more than 80% of the human proteome) in orchestrating different pathways in response to stimuli and triggering severe diseases, including cancer. NAA60/NatF (N-terminal acetyltransferase F), also named HAT4 (histone acetyltransferase type B protein 4), is a newly discovered acetyltransferase in humans modifying N-termini of transmembrane proteins starting with M–K/M-A/M-V/M-M residues and is also thought to modify lysine residues of histone H4. Because of its enzymatic features and unusual cell localization on the Golgi membrane, NAA60 is an intriguing acetyltransferase that warrants biochemical and clinical investigation. Although it is still poorly studied, this review summarizes current findings concerning the structural hallmarks and biological role of this novel targetable epigenetic enzyme.

BACKGROUND Transplantation of kidneys from hepatitis C virus (HCV)-viremic donors into HCV-negative patients followed by direct-acting antiviral therapy was an important breakthrough to increase the number of life-saving kidney transplants. Data suggest that these transplants offer several benefits; however, it is unknown whether adoption of this practice has been shared equitably, especially among disadvantaged groups. METHODS We evaluated United Network for Organ Sharing data on HCV-seronegative adult deceased-donor kidney transplant recipients from January 1, 2017, to June 12, 2020. We compared recipients of a kidney from an HCV antibody- (Ab-)/nucleic acid test- (NAT-), HCV Ab+/NAT-, and HCV NAT+ donor. The primary covariates were as follows: (1) race/ethnicity; (2) female sex; and (3) highest level of education. Models included variables associated with being offered an HCV NAT+ kidney. We fit mixed-effects multinomial logistic regression models with the center as a random effect to account for patient clustering. RESULTS Of 48 255 adult kidney-alone deceased-donor kidney transplant HCV-seronegative recipients, 1641 (3.4%) donors were HCV NAT+-, increasing from 0.3% (January 2017-June 2017) to 6.9% (January 2020-June 2020). In multivariable models, racial/ethnic minorities, women, and those with less education were significantly less likely to receive a kidney from an HCV NAT+ donor relative to an HCV Ab-/NAT- and HCV Ab+/NAT- donor. The disparities were most pronounced among Hispanic and Asian patients with less educational attainment (grade school, high school, or some college/tech school). CONCLUSIONS Despite an increase in transplants from HCV NAT+ donors, we found substantial racial/ethnic disparities in transplantation of these kidneys. These data highlight how the benefits of a scientific breakthrough are often made less available to disadvantaged patients.

Purpose: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. Experimental Design: We obtained pretreatment core-needle biopsies from 105 patients with stage I–III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT. Results: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 μm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. Conclusions: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

INTRODUCTION: Neoadjuvant therapy (NAT) is an emerging strategy for operable pancreatic ductal adenocarcinoma (PDAC). While neoadjuvant therapy increases multimodal therapy completion, it risks functional decline and treatment drop out. We used decision analysis to determine optimal management of localized PDAC and consider risks faced by elderly patients. METHODS: A Markov cohort decision analysis model evaluated treatment options for a 60-year-old patient with resectable PDAC: 1) upfront pancreaticoduodenectomy or 2) neoadjuvant therapy (NAT). One-way and probabilistic sensitivity analyses were performed. A sub-analysis considered the scenario of a 75-year-old patient. RESULTS: For the base case, NAT offered an incremental survival gain of 4.6 months compared to SF (overall survival: 26.3 vs 21.7 months). In One-way sensitivity analyses, findings were sensitive to recurrence free survival for NAT patients undergoing adjuvant, probability of completing NAT, and probability of being resectable at exploration after NAT. On probabilistic analysis, NAT was favored in a majority of trials (97%) with a median survival benefit of 5.1 months. In altering the base case for the 75-year-old scenario, NAT had a survival benefit of 3.8 months. CONCLUSIONS: This analysis demonstrates a significant benefit to neoadjuvant therapy in patients with localized PDAC. This benefit persists even in the elderly cohort.

Background Nucleic acid amplification testing (NAT) for blood screening has been previously performed in some countries to determine NAT yields. The current study sought to explore the non-discriminating reactive NAT yields using individual-NAT (ID-NAT) and characteristics of HBV NAT yields through a 10-year retrospective analysis in Zhejiang, China. Methods Blood donations were analyzed using individual-NAT mode by the transcription-mediated amplification (TMA) method. Supplementary HBV serological tests were performed using chemiluminescent immunoassay, and HBV viral load assay was performed by real-time polymerase chain reaction. Follow-up studies were performed in partial donors with low HBV viral loads. Results Non-discriminating reactive NAT yields and HBV NAT yields varied in different years. The yields ranged from 853.73 per million to 2018.68 per million and 624.60 per million to 1669.50 per million, respectively. In the 476 NAT yields, 19 were probable window periods (WP), 33 probable occult hepatitis B virus infections (OBIs), 409 were confirmed OBIs and 15 were chronic HBV infections. ID-NAT results were categorized in four groups, and the findings showed that the levels of HBV DNA viral loads were different in the four different groups (χ2 = 275.02, p 2 = 49.429, p Conclusion NAT screening of blood donations can reduce the risk of transfusion-transmitted HBV infections. Positive proportions of anti-HBs and anti-HBc are correlated with the HBV viral load level. However, low level of viral load donors pose risks in HBV NAT assays, and show fluctuating state for HBV viral load and leads to non-repeated NAT results during follow up studies.

International journal of molecular sciences 23(18), 10805 - (2022). doi:10.3390/ijms231810805, Most eukaryotic proteins are N-terminally acetylated by a set of N$\alpha$ acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi $Chaetomium$ $thermophilum$ and $Neurospora$ $crassa$ with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution., Published by Molecular Diversity Preservation International, Basel

Despite its increased application in pancreatic ductal adenocarcinoma (PDAC), complete response to neoadjuvant therapy (NAT) is rare. Given the critical role of host immunity in regulating cancer, we sought to correlate baseline inflammatory profiles to significant response to NAT. PDAC patients receiving NAT were classified as responders (R) or nonresponders (NR) by carbohydrate antigen 19-9 response, pathologic tumor size, and lymph node status in the resected specimen. Baseline (treatment-naive) plasma was analyzed to determine levels of 27 inflammatory mediators. Logistic regression was used to correlate individual mediators with response. Network analysis and Pearson correlation maps were derived to determine baseline inflammatory mediator profiles. Forty patients (20R and 20NR) met study criteria. The R showed significantly higher overall survival (59.4 vs. 21.25 mo, P=0.002) and disease-free survival (50.97 vs. 10.60 mo, P=0.005), compared with NR. soluble interleukin-2 receptor alpha was a significant predictor of no response to NAT (P=0.045). Analysis of inflammatory profiles using the Pearson heat map analysis followed by network analysis depicted increased inflammatory network complexity in NR compared with R (1.69 vs. 1), signifying a more robust baseline inflammatory status of NR. A panel of inflammatory mediators identified by logistic regression and Fischer score analysis was used to create a potential decision tree to predict NAT response. We demonstrate that baseline inflammatory profiles are associated with response to NAT in PDAC, and that an upregulated inflammatory status is associated with a poor response to NAT. Further analysis into the role of inflammatory mediators as predictors of chemotherapy response is warranted.

The role of lifestyle behaviors on neck adipose tissue (NAT), a fat depot that appears to be involved in the pathogenesis of different cardiometabolic diseases and in inflammatory status, is unknown. In this cross-sectional and exploratory study, the authors examined the relationship between sedentary time and physical activity (PA) with neck adiposity in young adults. A total of 134 subjects (69% women, 23 ± 2 years) were enrolled. The time spent in sedentary behavior and PA of different intensity were objectively measured for 7 consecutive days (24 hr/day), using a wrist (nondominant)-worn accelerometer. The NAT volume was assessed using computed tomography, and the compartmental (subcutaneous, intermuscular, and perivertebral) and total NAT volumes were determined at the level of vertebra C5. Anthropometric indicators and body composition (by dual-energy X-ray absorptiometry) were determined. The time spent in light physical activity and moderate physical activity (MPA) and the overall PA were inversely associated with the intermuscular NAT volume in men, as were the MPA and overall PA with total NAT volume (all ps ≤ .04). Sedentary time was directly related to the total NAT volume (p = .04). An opposite trend was observed in women, finding a direct relationship of MPA with the subcutaneous NAT; of light physical activity, MPA, and overall PA with the perivertebral NAT; and of light physical activity with total NAT volumes (all ps ≤ .05). The observed associations were weak, and after adjusting for multiplicity, the results became nonsignificant (p > .05). These findings suggest that the specific characteristics of PA (time and intensity) might have sex-dependent implications in the accumulation of NAT.

To compare overall survival (OS) in patients who underwent surgery for early-stage pancreatic adenocarcinoma (rPca) based on sequence (NAT, neoadjuvant therapy and/or AT, adjuvant therapy) and type (SA, single-agent or MA, multi-agent) of chemotherapy received.Using the National Cancer Database, patients with clinical stage I/II rPca diagnosed between 2010 and 2014 were identified and five comparison matches (1: NAT vs. upfront resection (UR); 2: multi-agent neoadjuvant (MA NAT) vs. single-agent adjuvant therapy (SA AT), single-agent neoadjuvant therapy (SA NAT), multi-agent adjuvant therapy (MA AT); 3: MA NAT vs. MA AT; 4: NAT + AT vs NAT; 5: NAT + AT vs AT) were constructed using minimum distance matching strategy. Median OS (mOS) was analysed using Kaplan-Meier method, log-rank test and Cox proportional hazard model.A total of 18,470 patients with stage I/II rPca were eligible for analysis. NAT showed a 5 month (mo.) improved OS compared with UR (3271 patients/group, 28.1 vs 23.2 mo. P 0.0001 hazard ratio [HR]: 0.79). MA-NAT was shown to be superior to other chemotherapy approaches SA AT, SA NAT, and MA AT (1349 patients/group: 30 vs. 25.9 mo., P = 0.0001 [HR: 0.82]). MA NAT showed a survival advantage over MA-AT (1349 patients/group, 30 vs 26.1 mo., P = 0.0008 [HR: 0.86]). The combination of NAT and AT showed a better outcome when compared with NAT alone (1128 patients/group, 31.6 vs 27.4 mo., P = 0.0011 [HR: 0.81]) or AT alone (1128 patients/group, 31.6 vs. 25.2 mo., P 0.0001 [HR: 0.76]).In patients with stage I/II rPca, MA NAT showed improved mOS compared to UR and all other chemotherapy sequences except both NAT plus AT. These findings support the use of MA NAT in stage I/II rPca patients and warrant prospective trials evaluating MA NAT and post-resection maintenance therapies.

Background Testing plays a critical role in treatment and prevention responses to the COVID-19 pandemic. Compared to nucleic acid tests (NATs), antigen-detection rapid diagnostic tests (Ag-RDTs) can be more accessible, but typically have lower sensitivity and specificity. By quantifying these trade-offs, we aimed to inform decisions about when an Ag-RDT would offer greater public health value than reliance on NAT. Methods Following an expert consultation, we selected two use cases for analysis: rapid identification of people with COVID-19 amongst patients admitted with respiratory symptoms in a ‘hospital’ setting and early identification and isolation of people with mildly symptomatic COVID-19 in a ‘community’ setting. Using decision analysis, we evaluated the health system cost and health impact (deaths averted and infectious days isolated) of an Ag-RDT-led strategy, compared to a strategy based on NAT and clinical judgement. We adopted a broad range of values for ‘contextual’ parameters relevant to a range of settings, including the availability of NAT and the performance of clinical judgement. We performed a multivariate sensitivity analysis to all of these parameters. Results In a hospital setting, an Ag-RDT-led strategy would avert more deaths than a NAT-based strategy, and at lower cost per death averted, when the sensitivity of clinical judgement is less than 90%, and when NAT results are available in time to inform clinical decision-making for less than 85% of patients. The use of an Ag-RDT is robustly supported in community settings, where it would avert more transmission at lower cost than relying on NAT alone, under a wide range of assumptions. Conclusions Despite their imperfect sensitivity and specificity, Ag-RDTs have the potential to be simultaneously more impactful, and have a lower cost per death and infectious person-days averted, than current approaches to COVID-19 diagnostic testing.

The ongoing pandemic of coronavirus disease 2019 threatens the whole world, which catalyzes a variety of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid test (NAT) kits. To monitor test quality and evaluate NAT kits, quality control materials that best simulate real clinical samples are needed. In this study, the performance of SARS-CoV-2 cell culture supernatant, PCDH-based pseudovirus, and MS2-based pseudovirus as quality control materials was compared. PCDH-based pseudovirus was found to be more similar in characteristics to SARS-CoV-2 particle, and more suitable for evaluating SARS-CoV-2 NAT kits than MS2-based pseudovirus. Proper detection using sensitive and precise NAT kits is essential to guarantee diagnosis. Thus, limit of detection, precision, anti-inference ability, and cross-reactivity of NAT kits from PerkinElmer, Beijing Wantai Biological Pharmacy Enterprise Co, Ltd, Shanghai Kehua Bio-Engineering Co, Ltd, Sansure Biotech Inc., Da An Gene Co, Ltd, Shanghai BioGerm Medical Biotechnology Co, Ltd, and Applied Biological Technologies Co, Ltd, were compared using PCDH-based pseudovirus. For the seven kits evaluated, N gene was more sensitive than ORF1ab gene in most kits, whereas E gene was most sensitive among the three genes in Shanghai Kehua Bio-Engineering Co, Ltd, and Applied Biological Technologies Co, Ltd. PerkinElmer got the lowest limit of detection for N gene at 11.61 copies/mL, and the value was 34.66 copies/mL for ORF1ab gene. All of the kits showed good precision, with CV values less than 5%, as well as acceptable anti-interference ability of 2 mg/L human genomic DNA. No cross-reactivity was observed with other respiratory viruses.

The actin cytoskeleton is of profound importance to cell shape, division, and intracellular force generation. Profilins bind to globular (G-)actin and regulate actin filament formation. Although profilins are well-established actin regulators, the distinct roles of the dominant profilin, profilin 1 (PFN1), versus the less abundant profilin 2 (PFN2) remain enigmatic. In this study, we use interaction proteomics to discover that PFN2 is an interaction partner of the actin N-terminal acetyltransferase NAA80, and further confirm this by analytical ultracentrifugation. Enzyme assays with NAA80 and different profilins demonstrate that PFN2 binding specifically increases the intrinsic catalytic activity of NAA80. NAA80 binds PFN2 through a proline-rich loop, deletion of which abrogates PFN2 binding. Small-angle X-ray scattering shows that NAA80, actin, and PFN2 form a ternary complex and that NAA80 has partly disordered regions in the N-terminus and the proline-rich loop, the latter of which is partly ordered upon PFN2 binding. Furthermore, binding of PFN2 to NAA80 via the proline-rich loop promotes binding between the globular domains of actin and NAA80, and thus acetylation of actin. However, the majority of cellular NAA80 is stably bound to PFN2 and not to actin, and we propose that this complex acetylates G-actin before it is incorporated into filaments. In conclusion, we reveal a functionally specific role of PFN2 as a stable interactor and regulator of the actin N-terminal acetyltransferase NAA80, and establish the modus operandi for NAA80-mediated actin N-terminal acetylation, a modification with a major impact on cytoskeletal dynamics. publishedVersion

Until recently, cancer registries have only collected cancer clinical stage at diagnosis, before any therapy, and pathological stage after surgical resection, provided no treatment has been given before the surgery, but they have not collected stage data after neoadjuvant therapy (NAT). Because NAT is increasingly being used to treat a variety of tumors, it has become important to make the distinction between both the clinical and the pathological assessment without NAT and the assessment after NAT to avoid any misunderstanding of the significance of the clinical and pathological findings. It also is important that cancer registries collect data after NAT to assess response and effectiveness of this treatment approach on a population basis. The prefix y is used to denote stage after NAT. Currently, cancer registries of the American College of Surgeons' Commission on Cancer only partially collect y stage data, and data on the clinical response to NAT (yc or posttherapy clinical information) are not collected or recorded in a standardized fashion. In addition to NAT, nonoperative management after radiation and chemotherapy is being used with increasing frequency in rectal cancer and may be expanded to other treatment sites. Using examples from breast, rectal, and esophageal cancers, the pathological and imaging changes seen after NAT are reviewed to demonstrate appropriate staging.

Background Postoperative pancreatic fistula (POPF) remains a prominent complication following pancreatic cancer resections. The primary aim of this study was to evaluate the histological changes that occur in the pancreas due to neoadjuvant therapy (NAT) by comparing the acinar, collagen and fat scores in resected PDAC specimens of patients who did and did not receive NAT. Secondary aims included (1) the difference in rates of POPF in PDAC patients who received NAT versus upfront resection; and (2) the association between acinar/collagen/fat scores and the development of POPF. Methods Consecutive patients who underwent pancreaticoduodenectomy for PDAC, with and without NAT were included for analysis. Acinar, collagen and fat scores were determined from histology slides of the pancreatic resection margin. Results One hundred and thirty-four patients were included. There was a significant decrease in the median acinar score (48 vs 23, p = 0.003) and increase in the collagen score (28 vs 50, p = 0.011) for patients who received NAT and a significant correlation with the number of cycles of NAT. This study found no statistical difference between NAT and the development of POPF. Conclusion The use of NAT in the treatment of PDAC changes the composition of the pancreas.

The kidney donor profile index (KDPI) defines an hepatitis C (HCV) positive donor based on HCV antibody (Ab) and/or nucleic acid amplification test (NAT) positivity, with donors who are not actively infected (Ab+/NAT-) also classified as HCV positive. From Scientific Registry of Transplant Recipients dataset, we identified HCV-negative recipients, who received a kidney transplant from HCV Ab+/NAT- (n = 116) and HCV Ab-/NAT- (n = 25 574) donor kidneys. We then compared recipients' estimated glomerular filtration rate (eGFR) at 6 months in matched cohorts, using combined exact matching (based on KDPI) and propensity score matching. We created two separate matched cohorts: for the first cohort, we used the allocation KDPI, while for the second cohort we used an optimal KDPI, where the HCV component of KDPI was considered negative in Ab+/NAT- patients. The mean ± SD age of the allocation KDPI-matched cohort at baseline was 59 ± 10 years, 69% were male, 61% were white. Recipients' eGFR at 6 months after transplantation was significantly higher in the HCV Ab+/NAT- group compared to the HCV Ab-/NAT- group (61.1 ± 17.9 vs. 55.6 ± 18.8 ml/min/1.73 m2 , P = 0.011) in the allocation KDPI-matched cohort, while it was similar (61.8 ± 19.5 vs. 62.1 ± 20.1 ml/min/1.73 m2 , P = 0.9) in the optimal KDPI-matched cohort. Recipients who received HCV Ab positive, but NAT-negative donor kidneys did not experience worse 6-month eGFR than correctly matched HCV Ab-/NAT- recipients.

At the time of diagnosis, only about 20% of patients with pancreatic ductal adenocarcinoma (PDAC) have resectable disease. PDAC treatment necessitates a multidisciplinary approach, and adjuvant chemotherapy after upfront resection is an established means of preventing recurrence. Neoadjuvant chemotherapy (NAT), originally introduced to downstage tumor size, is nowadays more frequently used for selection of patients with favorable tumor biology and to control potential micrometastases. While NAT is routinely applied in locally advanced (LA) PDAC, there is increasing evidence demonstrating benefits of NAT in borderline resectable (BR) PDAC. The concept of NAT has recently been tested in resectable PDAC, but to date NAT has been restricted to clinical trials, as the data are limited and no clear benefits have yet been shown in this patient group. This review summarizes the current evidence for NAT in resectable, BR, and LA PDAC, with a focus on high-level evidence and randomized controlled trials.

Neoadjuvant treatment (NAT) can downstage breast cancer and can be utilized for different clinical applications. However, the response to NAT varies among individuals. Having effective biomarkers is important to optimize the treatment of breast cancer. Concentrations of biogenic amines have been found to show an association with cancer cell proliferation, but their clinical utility remains unclear. This study developed a postcolumn-infused internal standard (PCI-IS)-assisted liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method for profiling biogenic amines in human urine. Putrescine-d8 was selected as the PCI-IS to calibrate the errors caused by matrix effects in the urine sample. The optimized method was applied to investigate the association between changes in 14 amines and the therapeutic response to NAT in breast cancer patients. Urine samples were collected before initiation of chemotherapy (n = 60). Our results indicated that the levels of N1-acetylspermine, spermidine, norepinephrine, and dopamine were significantly higher in the responder group than the nonresponder group. These metabolites were incorporated with clinical factors to identify NAT responders, and the prediction model showed an area under the curve value of 0.949. These observations provide remarkable insights for future studies in elucidating the roles of biogenic amines in breast cancer. Additionally, the PCI-IS-assisted amine profiling method can facilitate these studies.

Background Measuring incidence is important for monitoring and maintaining the safety of the blood supply. Blood collected from repeat-donors has provided the opportunity to follow blood donors over time and has been used to estimate the incidence of viral infections. These incidence estimates have been extrapolated to first-time donors using the ratio of NAT yield cases in first-time versus repeat-donors. We describe a model to estimate incidence in first-time donors using the limiting antigen (LAg) avidity assay and compare its results with those from established models. Methods HIV-positive first-time donations were tested for recency using the LAg assay. Three models were compared; incidence estimated for (1) first-time donors using LAg avidity, (2) first-time and repeat-donors separately using the NAT yield window period (WP) model and (3) repeat-donors using the incidence/WP model. Results HIV incidence in first-time donors was estimated at 3·32 (CI 3·11, 3·55) and 3·81 (CI 3·07, 4·73) per 1000 PY using the LAg assay and NAT yield WP models, respectively. Incidence in repeat-donors was between 2·0- and 2·5-fold lower than in first-time donors estimated at 1·56 (CI 1·37, 1·77) and 1·94 (CI 1·86-2·01) per 1000 PY using the NAT yield/WP and incidence/WP models, respectively. Conclusion Testing HIV-positive donations using the LAg assay provides a reliable method to estimate incidence in first-time donors for countries that collect the majority of blood from first-time donors and do not screen with NAT.

This study aimed to reassess the duration of neoadjuvant therapy (NAT) for patients with borderline resectable pancreatic cancer (BRPC). The medical records of patients with BRPC who received NAT before intended curative resection were retrospectively reviewed. Patient demographics, clinicopathological factors, and prognostic factors for overall survival were analyzed. The serum carbohydrate antigen (CA) 19-9 level was examined monthly during NAT. A total of 118 patients with BRPC were enrolled. The median survival time and 5-year overall survival were 28.0 months and 31%, respectively. Three months after NAT, the CA19-9 levels were normal in 57% of the patients, and 92% underwent resection. Multivariate analysis showed that radiological partial response (hazard ratio (HR), 0.53; 95% confidence interval (CI), 0.26–0.99; p = 0.047); a normal CA19-9 level after NAT (HR, 0.30; 95% CI, 0.22–0.66; p = 0.006); and tumor resection (HR, 0.29; 95% CI, 0.13–0.67; p = 0.005) were independent predictors of better survival. The median CA19-9 level and the rate of normal CA19-9 levels before and after NAT were 256 (interquartile range (IQR), 23–1197) U/mL and 33%, and 27 (IQR, 7–176) U/mL and 57%, respectively. A normal CA19-9 level after NAT was an independent predictor of better survival in patients with BRPC. A longer NAT duration might contribute to improved prognosis of patients with elevated CA19-9 levels.

Background Coronavirus disease-2019 (COVID-19) has spread widely throughout the world since the end of 2019. Nucleic acid testing (NAT) has played an important role in patient diagnosis and management of COVID-19. In some circumstances, thermal inactivation at 56°C has been recommended to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) before NAT. However, this procedure could theoretically disrupt nucleic acid integrity of this single-stranded RNA virus and cause false negatives in real-time polymerase chain reaction (RT-PCR) tests. Methods We investigated whether thermal inactivation could affect the results of viral NAT. We examined the effects of thermal inactivation on the quantitative RT-PCR results of SARS-CoV-2, particularly with regard to the rates of false-negative results for specimens carrying low viral loads. We additionally investigated the effects of different specimen types, sample preservation times, and a chemical inactivation approach on NAT. Results Our study showed increased Ct values in specimens from diagnosed COVID-19 patients in RT-PCR tests after thermal incubation. Moreover, about half of the weak-positive samples (7 of 15 samples, 46.7%) were RT-PCR negative after heat inactivation in at least one parallel testing. The use of guanidinium-based lysis for preservation of these specimens had a smaller impact on RT-PCR results with fewer false negatives (2 of 15 samples, 13.3%) and significantly less increase in Ct values than heat inactivation. Conclusion Thermal inactivation adversely affected the efficiency of RT-PCR for SARS-CoV-2 detection. Given the limited applicability associated with chemical inactivators, other approaches to ensure the overall protection of laboratory personnel need consideration.

Background and objectives Comparison of two models for estimating residual transfusion transmission risk by NAT screened window period (WP) donations in South African repeat donors gave identical results for HIV but not for HBV. In order to understand discrepant HBV modelling outcomes, the values of input parameters in three HBV WP risk models were reviewed and subsequently applied to the same South African screening data generated by HBsAg PRISM and two NAT assays (Ultrio and Ultrio Plus). Two of the models were also compared using individual donation (ID)-NAT screening data from different geographical regions. Methods Values of input parameters were derived from two published data sources and used in three risk models [(1) the incidence rate-WP risk day equivalent model, (2) the NAT yield WP ratio model and (3) the anti-HBc-negative HBsAg yield period ratio model] and subsequently applied to the same ID-NAT screening data. Results The HBV WP transmission risk in South African repeat donations during a one-year Ultrio Plus NAT screening period was estimated as 22, 43 and 17 per million, respectively, for the three models, as compared to 56, 117 and 48 per million for HBsAg PRISM screening. The approximate two-fold higher estimate calculated with the NAT yield WP ratio model was corroborated in repeat donations from three of four regions in a multi-regional study. When another set of model input values (with shorter viraemia periods and a higher proportion of acute occult infections) was applied to the South African screening data, the relative difference in risk estimates between the three models became smaller. Conclusions Window period risk modelling for HBV is more complex than for HIV. Multiple factors affect the modelling outcomes. These include the values used for the length of transient HBsAg and HBV-DNA-positive phases, the proportion of acute occult and vaccine breakthrough infections and the assumption of random appearance of donors throughout the entire acute resolving infection phase. A substantial proportion of HBV WP NAT yields have very low viral load and lack donor follow-up data calling into question their definitive classification into the early acute (infectious) replication stage. Since these possible WP NAT yields most highly impact the NAT yield WP ratio model, we recommend relying on the more conservative estimates of the incidence rate-WP risk day equivalent model.

Background West Nile Virus (WNV) is a member of the Japanese Encephalitis (JE) serocomplex within the Flaviviridae family. We report four whole blood donors and one plasma donor with WNV nucleic acid test (NAT)-reactive donations between September 2018 and November 2019, following recent Japanese Encephalitis virus (JEV) vaccination. Case series Cases 1 and 4 had reactive WNV NAT donations 1 day after receiving the JEV vaccine. Case 2 had a reactive WNV donation 3 days after receiving the JEV vaccine. Case 3 had a reactive WNV NAT donation 3 days after returning from Arizona and 1 day after receiving the JEV vaccine. Case 5 had a reactive WNV donation the same day as receiving the JEV vaccine. Study design and methods WNV screening used the Roche cobas WNV nucleic acid test (NAT) (Roche Molecular Systems). Reference testing on WNV-reactive donations was carried out by the National Microbiology Laboratory (NML). JEV vaccine dilutions were also analyzed. Results Supplemental NAT was negative for WNV and JEV for Cases 1, 3, and 5. Case 2 had a weak amplification curve for one of two JEV NAT targets. Case 4 was JEV NAT-positive, WNV NAT-negative. Serologic testing on donation specimens for Cases 2, 4, and 5 did not support recent or remote WNV infection. JEV vaccine dilutions were detected by both cobas and supplemental NAT. Conclusions We recommend implementing a temporary blood donor deferral following a JEV vaccination, if screening utilizes a WNV assay with the capability of detecting other members of the JE serocomplex.

Background Pediatric nonaccidental trauma (NAT) accounts for more than 1500 deaths annually and is a source of incalculable lifelong morbidity. Evidence-based NAT evaluation protocols are available; however, compliance studies are lacking. Here, we analyze the quality of implementation of a new NAT evaluation protocol. Methods A review of registry data from a level 3 trauma center was performed for patients with a suspicion of NAT from September 2014 to May 2016. Compliance rates and results of 2 new age-based evaluation protocols were examined before (phase 1) and after (phase 2) a multidisciplinary conference was initiated to improve performance. Results Seventy-five children underwent evaluation for NAT during the study period. In phase 1, median compliance rates with all of the protocol elements were low (63%). After a bimonthly multidisciplinary conference was initiated, compliance rates improved to 75%. Some elements, which were often missed, were found to be clinically irrelevant. If only clinically relevant elements were included, median compliance in phase 2 is 86%. Conclusions Compliance with a new protocol for NAT was improved with the institution of a multidisciplinary conference. Protocol elements, which are shown to be clinically unnecessary, have also been identified and will be eliminated.

Background: Hepatitis B virus infection is one of the greatest threats to blood safety all over the world. The laboratory algorithm based on only the detection of hepatitis B surface antigen (HBsAg) leaves a gap for infected HBsAg negative donors to donate blood during the “window period” (WP) and late stages of infection. Objective: To estimate the frequency of the presence of HBV deoxyribonucleic acid (DNA) in HBsAg negative blood units screened using two different assays for HBsAg in a high endemic region. Methods: Frozen serum aliquot of 100 replacement blood donors who donated blood units that were HBsAg negative were retrieved and tested for HBV DNA. Sample positive for HBV DNA was sequenced by Sanger’s method, genotyped and the viral load was determined. Results: One sample (1%) was positive for HBV DNA. The HBV viral load of the sample was 768,000 IU/ml. The partial S-gene of the Hepatitis B virus isolated was genotype E using the NCBI viral genotyping tool. Conclusions: There is still a risk of HBV infected blood unit escaping detection when donor testing is limited to HBsAg screening. The use of NAT which can substantially reduce HBV infected blood donors from the donor pool should be considered. Keywords: Hepatitis B surface Antigen; Hepatitis B Virus; DNA; blood donors; blood safety.

On April 2021, the United States Organ Procurement and Transplantation Executive Committee approved the "Lower Respiratory SARS-CoV-2 Testing for Lung Donors" emergency policy upon recommendation from the Ad Hoc Disease Transmission Advisory Committee. This policy requires that all lung donors be tested for SARS-CoV-2 in a lower respiratory specimen by nucleic acid test and that the results be available before the lungs are transplanted. The overarching goal of the emergency policy was to minimize the risk of donor-derived COVID-19 to lung recipients. However, an unintended consequence of the policy was the emergence of a new population of potential donors: the SARS-CoV-2 lower respiratory tract nucleic acid test positive donor. We describe the use of two SARS-CoV-2 lower respiratory tract nucleic acid test positive liver donors without a known history of COVID-19 infection with adequate short-term outcomes. The recipients did not have a prior history of COVID-19, nor did they receive monoclonal antibodies post-transplantation; one was unvaccinated. If the safety and long-term outcomes from SARS-CoV-2 LRT NAT positive donors is confirmed in larger studies, this strategy represents a promising way to increase the pool for organ donation. This article is protected by copyright. All rights reserved.

Background The purpose of this study was to determine whether advanced quantitative magnetic resonance imaging (MRI) can be deployed outside of large, research-oriented academic hospitals and into community care settings to predict eventual pathological complete response (pCR) to neoadjuvant therapy (NAT) in patients with locally advanced breast cancer. Methods Patients with stage II/III breast cancer (N = 28) were enrolled in a multicenter study performed in community radiology settings. Dynamic contrast-enhanced (DCE) and diffusion-weighted (DW)-MRI data were acquired at four time points during the course of NAT. Estimates of the vascular perfusion and permeability, as assessed by the volume transfer rate (Ktrans) using the Patlak model, were generated from the DCE-MRI data while estimates of cell density, as assessed by the apparent diffusion coefficient (ADC), were calculated from DW-MRI data. Tumor volume was calculated using semi-automatic segmentation and combined with Ktrans and ADC to yield bulk tumor blood flow and cellularity, respectively. The percent change in quantitative parameters at each MRI scan was calculated and compared to pathological response at the time of surgery. The predictive accuracy of each MRI parameter at different time points was quantified using receiver operating characteristic curves. Results Tumor size and quantitative MRI parameters were similar at baseline between groups that achieved pCR (n = 8) and those that did not (n = 20). Patients achieving a pCR had a larger decline in volume and cellularity than those who did not achieve pCR after one cycle of NAT (p Ktrans, ADC, cellularity, and bulk tumor flow from baseline (pre-treatment) were all significantly greater (p Conclusions Quantitative analysis of DCE-MRI and DW-MRI can be implemented in the community care setting to accurately predict the response of breast cancer to NAT. Dissemination of quantitative MRI into the community setting allows for the incorporation of these parameters into the standard of care and increases the number of clinical community sites able to participate in novel drug trials that require quantitative MRI.

One of the grand challenges for field-deployable NATs is related to the front end of the assays – nucleic acid extraction from raw samples. The ideal nucleic acid sample preparation should be simple, scalable, and easy-to-operate. In this chapter, we present a lab-on-a-disc NAT device for sample-to-answer malaria diagnosis. The parasite DNA sample preparation and subsequent real-time LAMP detection were seamlessly integrated on a disposable single microfluidic compact disc, driven by energy-efficient, non-centrifuge based magnetic field interactions. Each disc contains four parallel testing units, which could be configured either as four identical tests or as four species-specific tests. When configured as species-specific tests, it could identify two of the most life-threatening malaria species (P. falciparum and P. vivax). The reagent disc with a 4-plex analyzer (discussed in Chapter 1) is capable of processing four samples simultaneously with 40 min turnaround time. It achieves a detection limit of ~0.5 parasites/μl for whole blood, sufficient for detecting asymptomatic parasite carriers. The assay is performed with an automated device described in the companion chapter. The combination of sensitivity, specificity, cost, and scalable sample preparation suggests the real-time fluorescence LAMP device could be particularly useful for malaria screening in the field settings.

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT.

Hepatitis B virus (HBV) infection is the most frequent viral infection found in blood donors (BDs) in France. We analyzed the epidemiological and sero-molecular data on HBV infection gathered over the past two decades by the French haemovigilance surveillance network, blood screening laboratories, and the national reference center for transfusion infectious risks (NRC). Between 2000 and 2020, 6149 of the 58,160,984 donations (1.06/10,000) tested HBV positive, 98% of them from first-time blood donors (FTBDs). In addition, 2212 (0.0071%) of the 30,977,753 donations screened for HBV DNA tested DNA positive, of which 25 (1.1%) were positive only for this marker. HBV prevalence decreased by 2.8-fold and the residual risk for transfusion-transmitted HBV infection decreased 13-fold and was divided by 13. The major risk factor for HBV infection was the origin of donors (endemic country, 66.5%), followed by parenteral exposure (10.7%). In the whole HBV-positive BD population, genotype D was predominant (41.8%), followed by genotypes A (26.2%) and E (20.4%), reflecting the geographical origin of donors. The low and decreasing prevalence and incidence of HBV infection in French BDs, coupled with a screening strategy using three HBV markers (HBsAg, anti-HBc and DNA), ensures a high level of blood safety, further reinforced by the implementation of pathogen-reduction measures.

Objectives The aim of this study was to assess the 1-year safety and effectiveness of HBV Nucleic Acid Test positive (HBV NAT+) allografts in seronegative kidney transplant (KT) and liver transplant (LT) recipients. Summary background data Despite an ongoing organ shortage, the utilization of HBV NAT+ allografts into seronegative recipients has not been investigated. Methods From January 2017 to October 2020, a prospective cohort study was conducted among consecutive KT and LT recipients at a single institution. Primary endpoints were post-transplant HBV viremia, graft and patient survival. Results With median follow-up of 1-year, there were no HBV-related complications in the 89 HBV NAT+ recipients. Only 9 of 56 KTs (16.1%) and 9 of 33 LTs (27.3%) experienced post-transplant HBV viremia at a median of 185 (KT) and 269 (LT) days postoperatively. Overall, viremic episodes resolved to undetected HBV DNA after a median of 80 days of entecavir therapy in 16 of 18 recipients. Presently, 100% of KT recipients and 93.9% of LT recipients are HBV NAT- with median follow-up of 13 months, whereas 0 KT and 8 LT (24.2%) recipients are HBV surface antigen positive indicating chronic infection. KT and LT patient and allograft survival were not different between HBV NAT+ and HBV NAT- recipients (P > 0.05), whereas HBV NAT+ KT recipients had decreased waitlist time and pretransplant duration on dialysis (P Conclusions This is the largest series describing the transplantation of HBV NAT+ kidney and liver allografts into HBV seronegative recipients without chronic HBV viremia or decreased 1-year patient and graft survival. Increasing the utilization of HBV NAT+ organs in nonviremic recipients can play a role in decreasing the national organ shortage.

Background/Aim: Chloride intracellular channel 4 (CLIC4) is associated with the progression of colorectal cancer (CRC). However, quantitative differences in CLIC4 expression in epithelial and stromal cells of normal mucosal tissue (NT), cancer adjacent to normal colorectal mucosal tissue (NAT), and CRC tissue remain unclear. Materials and Methods: We investigated the number of CLIC4 high-expressing (CLIC4(high)) cells in colorectal tissue of CRC patients and healthy individuals. Results: The number of CLIC4(high) cells in malignant epithelial cells at early cancerous lesions was significantly higher than that in NAT, but was significantly lower or tended to become low corresponding to the progression of colorectal carcinogenesis. Meanwhile, the number of CLIC4(high) cells in the stromal tissue remained low in NAT compared to late lesions. Conclusion: The number of CLIC4(high) cells is a useful predictor in determining the pathological condition in both malignant epithelial and stromal tissues of CRC patients.

BackgroundMost Chinese Blood Centers adopted mini pool (MP) nucleic acid testing (NAT) for HBV screening due to high cost of Individual donation (ID) NAT, and different proportions of MP-reactive but ID-non-reactive donations (MP+/ID−, defined as non-resolved donations) have been observed during daily donor screening process. Some of these non-resolved donations are occult HBV infections (OBIs), which pose potential risk of HBV transmission if they are not deferred. This study is aimed to further analyze these non-resolved donations.MethodsThe non-resolved plasma samples were further analyzed by serological tests and various HBV DNA amplification assays including quantitative PCR (qPCR) and nested PCR amplifying the basic core and pre-core promoter regions (BCP/PC; 295 base pairs) and HBsAg (S) region (496 base pairs). Molecular characterizations of HBV DNA+ non-resolved samples were determined by sequencing analysis.ResultsOf 17,226 MPs from 103,356 seronegative blood donations, 98 MPs were detected reactive for HBV. Fifty-six out of these 98 (57.1%) reactive MPs were resolved as HBV DNA+, but the remaining 42 pools (42.9%, 252 donations) were left non-resolved with a high rate (53.2%) of anti-HBc+. Surprisingly, among 42 non-resolved MPs, 17 contained one donation identified as OBIs by alternative NAT assays. Sequence analysis on HBV DNAs extracted from these OBI donations showed some key mutations in the S region that may lead to failure in HBsAg detection and vaccine escape.ConclusionA total of 53.2% of the non-resolved donations were anti-HBc+, and OBIs were identified in 40.5% of these non-resolved pools. Therefore, non-resolved donations with anti-HBc+ might pose potential risk for HBV transmission. Our present analysis indicates that anti-HBc testing in non-resolved donations should be used to identify OBIs in order to further increase blood safety in China.

Nucleic acid testing (NAT) was implemented in Poland in 1999 for screening of plasma for fractionation and for all blood donors in 2002. To analyze seronegative NAT‐positive samples representing hepatitis C virus (HCV) window‐period (WP) in the years 2000 to 2016 and to determine infection outcome. We analyzed results of 17 502 739 donations screened in minipools (6‐48) or individually. Index samples underwent viral load (VL) quantification, genotyping and Ag, and anti‐HCV re‐testing using chemiluminescence (CMIA), electrochemiluminescence (ECLIA), and fourth‐generation enzyme‐linked immunosorbent assay (IV EIA) assays. HCV‐seronegative infections were identified in 126 donations (7.2/mln donations; 95% confidential intervals, 6.0‐8.6). Frequency of NAT yields was decreasing over time. Of the initial 126 seronegative index cases 106 were retested: 32.1% were reactive in IV EIA, 11.3% in ECLIA, and 1.9% in CMIA. The lowest VL correlated with absent anti‐HCV and HCV Ag, while VL was highest when the antigen was detectable and then it decreased when anti‐HCV appeared at a level detectable by sensitive third generation tests while retesting. The proportion of genotype 1 was 38.9% in samples positive only for HCV RNA and 71.4% in samples that were anti‐HCV reactive in re‐testing. In parallel, genotype 3 frequency was 50% in the former group and 21% in the latter. NAT is an effective measure to limit HCV transmission by transfusion and IV EIA seems to have higher clinical sensitivity than ECLIA. Samples representing likely successive phases of early HCV infection were characterized by different genotype distribution probably due to very early elimination of genotype 3., Highlight NAT is highly effective in prevention of transmission of HCV seronegative infections.In nearly 7% of followed up donors antibodies appeared later than 50 days after index donation.The highest clinical sensitivity was demonstrated by the IVth generation tests.Among the third generation assays, ECLIA showed higher sensitivity than CMIA.Samples representing likely successive phases of early HCV infection demonstrated different genotype distribution.

This study aimed to explore the prognostic significance of the resection margin (R) status of pancreatic ductal adenocarcinoma (PDAC) patients receiving neoadjuvant therapy (NAT) or adjuvant chemotherapy (AC). We retrospectively reviewed 427 consecutive patients, and the overall survival (OS) and disease-free survival (DFS) were analyzed based on the R status by a propensity score analysis (PSA). The R0 ratio of the NAT (+) group was significantly higher than that of the NAT (−) group (97.2% vs. 69.6%, P

The emergence of Zika virus (ZIKV) in the Americas has resulted in increased nucleic acid amplification testing (NAT) of clinical samples and blood donations. New molecular diagnostic assays have been developed resulting in a corollary requirement for ZIKV reference material. To address this we have produced and calibrated two African lineage ZIKV reference materials: a highly concentrated secondary standard (NIBSC: 16/110) and a lower concentration external quality control (QC) reagent (NIBSC: 16/124) and compared their performance in three ZIKV NAT assays in relation with the First International Standard (IS) for Zika Virus NAT assays (PEI: 11468/16). In summary the African lineage ZIKV reference materials were detected by all three assays. The ZIKV lineage did not affect the performance of the secondary standard. The external QC reagent (16/124) was detected by all three assays highlighting its suitability for use as a low positive control to monitor assay performance on a regular basis. The relative potency of 16/110 to the IS was 5.49E+06IU/mL (95% CI: 1.46E+06–2.06E+07) and 16/124 to 16/110 was 8.36E+03 (95% CI: 7.83E+03–8.92E+03). The global availability of African lineage ZIKV reference materials will facilitate standardization of ZIKV molecular diagnostic assays between and within laboratories whilst preserving the IS.

Recently, it has been discovered that different DNA-(cytosine C5)-methyltransferases including DNMT3A generate low levels of 3mC [Rosic et al. (2018), Nat. Genet., 50, 452–459]. This reaction resulted in the co-evolution of DNMTs and ALKB2 DNA repair enzymes, but its mechanism remained elusive. Here, we investigated the catalytic mechanism of DNMT3A for cytosine N3 methylation. We generated several DNMT3A variants with mutated catalytic residues and measured their activities in 5mC and 3mC generation by liquid chromatography linked to tandem mass spectrometry. Our data suggest that the methylation of N3 instead of C5 is caused by an inverted binding of the flipped cytosine target base into the active-site pocket of the DNA methyltransferase, which is partially compatible with the arrangement of catalytic amino acid residues. Given that all DNA-(cytosine C5)-methyltransferases have a common catalytic mechanism, it is likely that other enzymes of this class generate 3mC following the same mechanism.

Introduction Hepatitis B viral infection has been transmitted from donors with HBV infections who have negative HBs Ag. Many countries have implemented nucleic acid testing (NAT) to screen donors with non- reactive HBs Ag for detection of HBV DNA and enhance safety of blood transfusion, while it is restricted to limited blood banks in Egypt. Objective To evaluate the significance of NAT technology in detection of HBV DNA in the Egyptian blood donors with HBs Ag non- reactivity. Methods The study included 36,584 collected blood samples from volunteer blood donors at the blood bank of Zagazig University Hospitals. Each specimen was tested for HBs Ag; non- reactive sera were further tested for qualitative detection of HBV-DNA by NAT testing. All positive HBV-DNA donors were tested for anti- HBc and anti- HBs by electro-chemiluminescence immunoassay and confirmed by quantitative RT-PCR. Results Among 34,671 donors non-reactive to HBs Ag, 34,657 (99.96%) were tested negative for HBV- DNA and 14 specimens (0.04%) were positive for HBV via NAT testing. Among HBV NAT positive donors, HBs Ab reactive only in (2); HBc Ab reactive only in (3); HBs and HBc Abs reactive in (3) while HBs and HBc Ab non-reactive in (6). All tested sera 14 (100%) showed low viral load for HBV ( Conclusion Our results highlighted the significance of the HBV NAT technique to reduce the potential risk of HBV transfusion-transmission and the critical need to enforce the usage of NAT technology in all blood banks in Egypt.

The feasibility of neoadjuvant therapy (NAT) for elderly patients with rectal cancer has not been evaluated well. Between 2004 and 2014, 506 patients with locally advanced low rectal cancer underwent curative resection. Fifty-four were over 75 years old (elderly group), and 452 were under 75 years old (young group). The patients were divided into sub-groups according to whether they received NAT. Nineteen (35.2%) patients from the elderly group and 348 (77.0%) from the young group received NAT. The proportion of patients who received NAT was significantly lower in the elderly group. In the elderly group, the median age and prevalence of co-morbidities were significantly lower in patients with than in those without NAT. The incidence of severe adverse events was similar in the two groups. On multivariate analysis, age was not related to postoperative complications in patients who received NAT. The 5-year local recurrence rate was significantly lower in the elderly patients who received NAT, and similar to that of the young patients who received NAT. Neoadjuvant therapy was feasible and should be considered as a treatment option for carefully selected elderly patients with locally advanced low rectal cancer.

International audience; Recent studies have revealed a possible link between the activities of polymorphic arylamine N-acetyltransferases (NATs) and energy metabolism. We used a Nat1/Nat2 double knockout (KO) mouse model to demonstrate that ablation of the two Nat genes is associated with modest, intermittent alterations in respiratory exchange rate. Pyruvate tolerance tests show that double KO mice have attenuated hepatic gluconeogenesis when maintained on a high-fat/high-sucrose diet. Absence of the two Nat genes also leads to an increase in the hepatic concentration of coenzyme A in mice fed a high-fat/high-sucrose diet. Our results suggest a modest involvement of NAT in energy metabolism in mice, which is consistent with the absence of major phenotypic deregulation of energy metabolism in slow human acetylators.

Cytomegalovirus (CMV) is an opportunistic pathogen, and careful monitoring of CMV is important for immunocompromised patients. Antigenemia-based CMV monitoring is a standard test used for managing CMV infection in transplant recipients;however, in Japan, there are no reports of CMV monitoring using the standardized test. The utility of a standardized CMV nucleic acid test (NAT) was evaluated during antigenemia-based CMV monitoring after hematopoietic stem cell transplantation (HSCT) or liver transplantation. Blood collection for CMV monitoring was performed under the physician's instructions depending on the condition of the patient, and CMV NAT and antigenemia was evaluated. For HSCT recipients, blood collection only for NAT was additionally performed during the pre-engraftment phase. The results of the NAT were blinded to those evaluating the results. A total of 34 patients were enrolled (11 HSCT recipients and 23 liver transplant recipients). NAT detected the first CMV episode no later than antigenemia in 2 (18.2%) HSCT recipients and 3 (13.0%) liver transplant recipients, earlier than antigenemia in 3 (27.3%) HSCT recipients and 7 (30.4%) liver transplant recipients, and later than antigenemia in 1 (9.1%) HSCT recipient and 1 (4.3%) liver transplant recipient. In 5 HSCT recipients, NAT was positive during the pre-engraftment phase. Among the 468 blood samples which were evaluated by both NAT and antigenemia, 124 (26.7%) were positive in NAT and 51 (10.9%) were positive in antigenemia. The standardized CMV NAT is useful for accurately diagnosing CMV infection and determining appropriate therapeutic interventions for HSCT recipients and liver transplant recipients., The Tohoku journal of experimental medicine, 247(3), pp.179-187; 2019

Background & objectives: Nucleic acid amplification test (NAT) in blood donor screening not only detects window period (WP) donors but also those with chronic occult infections which are negative by routine serological screening. This study was conducted to determine the time trend of NAT positivity and seroprevalence of transfusion-transmitted infections (TTIs) through a period of six years and evaluate the strength of NAT as a supplementary test in identifying the cryptic carriers in blood donor population. Methods: A total of 1,01,411 blood donations were screened between January 2011 and December 2016 by the ELISA and individual donor (ID) NAT Procleix Ultrio Plus Assay. Additional molecular and serological assays were done on the NAT yield samples to differentiate the type of cryptic carriers. Results: NAT yields comprised 0.05 per cent (50/101411) of the total samples tested with a yield rate of 1/2028. Hepatitis B virus (HBV) contributed to 80 per cent of the total NAT yields and the rest 20 per cent due to hepatitis C virus (HCV). Majority of HBV NAT yields (75%) were from chronic occult donors and 25 per cent were WP donors. Both HBV and HCV NAT yields had a wide range of viral count. There was no HIV NAT yield. A significant decline in the prevalence rate of TTIs through the study period of six years was observed. Interpretation & conclusions: The cryptic infections found in blood donors increase the risk of TTIs. Blood screening by both serology and NAT can reduce this threat.

The use of allografts from hepatitis C virus (HCV) Nucleic Acid Testing (NAT)+ donors into HCV NAT- recipients has been reported to be efficacious in a handful of studies. However, these studies have not reflected real-world practice where the initiation of direct-acting antivirals (DAA) is dependent on insurance coverage. A single-center, retrospective chart review of HCV NAT- recipients who underwent solid organ transplantation (SOT) from a HCV NAT+ donor between April 1, 2019 and May 27, 2020 was conducted. Sixty-one HCV NAT- patients underwent SOT with a HCV NAT+ organ, with 59 transplant recipients included for evaluation: 22 kidney (KT), 18 liver (LiT), 10 heart (HT), nine lung (LuT). HCV transmission occurred in 100% of recipients. Average time to DAA initiation was POD 46.3 ± 25 days. SVR12 was achieved in 98% (56/57; two patients ineligible for analysis). Treatment failure occurred in one LuT on glecaprevir/pibrentasvir with P32del and Q80K mutations. No patients developed fibrosing cholestatic hepatitis. Two patients died, secondary to anastomotic complication (LuT) and pulmonary embolism (HT). Clinically significant rejection was diagnosed and treated in two HT (one patient with ACR2 and one with ACR2/pAMR2) and one LiT (RAI 5/9). Six patients (10.2%) had documented adverse effects attributed to DAA therapy, primarily gastrointestinal.

Background/aim In cases where neoadjuvant treatment (NAT) is administered, research on short-term postoperative outcomes appears to be insufficient. We compared short-term outcomes of upfront surgery (UpS) cases and NAT cases for pancreatic ductal adenocarcinoma (PDAC). Patients and methods We retrospectively reviewed 1,228 cases that had elective pancreatectomy at Samsung Medical Center from 2010 to 2020. All cases were classified into resectable pancreatic cancer (RPC) and locally advanced pancreatic cancer (LAPC) according to NCCN guidelines 2017. In each group, factors were compared between the UpS and NAT groups. Results Rate of vascular resection was higher in the NAT group in RPC, compared to that in the NAT group in LAPC. Short-term postoperative outcomes had no significant differences between the UpS and NAT groups in both RPC and LAPC. Conclusion In the NAT group, there were no significant differences from UpS in terms of short-term postoperative outcomes. Conversion surgery following NAT is a favorable strategy.