Southern Region Produce Safety Alliance Grower Training: Using Pre- and Post-Training Knowledge Assessments to Understand Training Effectiveness.

The Produce Safety Alliance (PSA) grower training was introduced in 2016 as the standardized curriculum to meet the training requirements of the Food and Drug Administration's (FDA) Food Safety Modernization Act's (FSMA) Produce Safety Rule (PSR). The PSR states that at least one supervisor or responsible party from each farm must have successfully completed this food safety training or one equivalent to the standardized curriculum, as recognized by the FDA. This study evaluated the effectiveness of PSA trainings conducted between 2017 and 2019 in the Southern United States by the Southern Regional Center for Food Safety Training, Outreach, and Technical Assistance by analyzing pre- and posttest assessments. Effectiveness was based on a 25-question knowledge assessment administered to participants before (n = 2494) and after (n = 2460) each training. The knowledge assessment indicated the overall effectiveness of the training, with average scores increasing significantly from pretest (15.9/25, 63.4%) to posttest (20.3/25, 81.3%) (P < 0.001). The greatest knowledge gains were seen in the Postharvest Handling and Sanitation, How to Develop a Farm Food Safety Plan, and Agricultural Water modules. Notably, these modules had lower posttest scores compared to the other modules, indicating that the amount of knowledge gained did not necessarily correspond with a sufficient understanding of the material. To ensure that participants understand all aspects of the PSR and best practices to minimize food safety risks, additional or advanced trainings may be needed. Additionally, the current testing instrument (pre-/posttest) used for PSA grower training, while validated, may not be optimal, thus alternative methods to assess the training effectiveness are likely needed.

The exonuclease TREX1 constitutes an innate immune checkpoint limiting cGAS/STING-mediated antitumor immunity.

The DNA exonuclease TREX1 (Three-prime repair exonuclease 1) is critical for preventing autoimmunity in mice and humans by degrading endogenous cytosolic DNA, which otherwise triggers activation of the innate cGAS/STING pathway leading to the production of type I IFNs. Since tumor cells are prone to aberrant cytosolic DNA accumulation, we hypothesized that they are critically dependent on TREX1 activity to limit their immunogenicity. Here we show, that in tumor cells TREX1 indeed restricts the spontaneous activation of the cGAS/STING pathway and the subsequent induction of a type I IFN response. As a result, TREX1 deficiency compromised in vivo tumor growth in mice. This delay depended on a functional immune system, systemic type I IFN signaling, and tumor-intrinsic cGAS expression. Mechanistically, we show that tumor TREX1 loss drove activation of CD8 T cells and NK cells, prevented CD8 T cell exhaustion, and remodeled an immunosuppressive myeloid compartment. Consequently, TREX1 deficiency synergized with T cell-directed immune checkpoint blockade. Collectively, we conclude that TREX1 is essential to limit tumor immunogenicity, and that targeting this innate immune checkpoint remodels the tumor microenvironment and enhances anti-tumor immunity by itself and in combination with T cell-targeted therapies.

Detection of Salmonella enterica and Listeria monocytogenes in alternative irrigation water by culture and qPCR-based methods in the Mid-Atlantic U.S.

Alternative irrigation waters (rivers, ponds, and reclaimed water) can harbor bacterial foodborne pathogens like Salmonella enterica and Listeria monocytogenes, potentially contaminating fruit and vegetable commodities. Detecting foodborne pathogens using qPCR-based methods may accelerate testing methods and procedures compared to culture-based methods. This study compared detection of S. enterica and L. monocytogenes by qPCR (real-time PCR) and culture methods in irrigation waters to determine the influence of water type (river, pond, and reclaimed water), season (winter, spring, summer, and fall), or volume (0.1, 1, and 10 L) on sensitivity, accuracy, specificity, and positive (PPV), and negative (NPV) predictive values of these methods. Water samples were collected by filtration through modified Moore swabs (MMS) over a 2-year period at 11 sites in the Mid-Atlantic U.S. on a bi-weekly or monthly schedule. For qPCR, bacterial DNA from culture-enriched samples (n = 1,990) was analyzed by multiplex qPCR specific for S. enterica and L. monocytogenes. For culture detection, enriched samples were selectively enriched, isolated, and PCR confirmed. PPVs for qPCR detection of S. enterica and L. monocytogenes were 68% and 67%, respectively. The NPV were 87% (S. enterica) and 85% (L. monocytogenes). Higher levels of qPCR/culture agreement were observed in spring and summer compared to fall and winter for S. enterica; for L. monocytogenes, lower levels of agreement were observed in winter compared to spring, summer, and fall. Reclaimed and pond water supported higher levels of qPCR/culture agreement compared to river water for both S. enterica and L. monocytogenes, indicating that water type may influence the agreement of these results. IMPORTANCE: Detecting foodborne pathogens in irrigation water can inform interventions and management strategies to reduce risk of contamination and illness associated with fresh and fresh-cut fruits and vegetables. The use of non-culture methods like qPCR has the potential to accelerate the testing process. Results indicated that pond and reclaimed water showed higher levels of agreement between culture and qPCR methods than river water, perhaps due to specific physiochemical characteristics of the water. These findings also show that season and sample volume affect the agreement of qPCR and culture results. Overall, qPCR methods could be more confidently utilized to determine the absence of Salmonella enterica and Listeria monocytogenes in irrigation water samples examined in this study.

Combined PD-L1/TGFß blockade allows expansion and differentiation of stem cell-like CD8 T cells in immune excluded tumors.

TGFß signaling is associated with non-response to immune checkpoint blockade in patients with advanced cancers, particularly in the immune-excluded phenotype. While previous work demonstrates that converting tumors from excluded to inflamed phenotypes requires attenuation of PD-L1 and TGFß signaling, the underlying cellular mechanisms remain unclear. Here, we show that TGFß and PD-L1 restrain intratumoral stem cell-like CD8 T cell (TSCL) expansion and replacement of progenitor-exhausted and dysfunctional CD8 T cells with non-exhausted T effector cells in the EMT6 tumor model in female mice. Upon combined TGFß/PD-L1 blockade IFNγhi CD8 T effector cells show enhanced motility and accumulate in the tumor. Ensuing IFNγ signaling transforms myeloid, stromal, and tumor niches to yield an immune-supportive ecosystem. Blocking IFNγ abolishes the anti-PD-L1/anti-TGFß therapy efficacy. Our data suggest that TGFß works with PD-L1 to prevent TSCL expansion and replacement of exhausted CD8 T cells, thereby maintaining the T cell compartment in a dysfunctional state.

Impact of insurance status on healthcare resource utilization and outcomes in adolescent patients presenting with spinal cord injuries.

OBJECTIVE: Insurance disparities have been suggested to influence the medical and surgical outcomes of adult patients with spinal cord injury (SCI), with a paucity of studies demonstrating their impact on the outcomes of pediatric and adolescent SCI patients. The aim of this study was to assess the impact of insurance status on healthcare utilization and outcomes in adolescent patients presenting with SCI. METHODS: An administrative database study was performed using the 2017 admission year from 753 facilities using the National Trauma Data Bank. Adolescent patients (11-17 years old) with cervical/thoracic SCIs were identified using International Classification of Diseases, Tenth Revision, Clinical Modification coding. Patients were categorized by governmental insurance versus private insurance/self-pay. Patient demographics, comorbidities, imaging, procedures, hospital adverse events (AEs), and length of stay (LOS) data were collected. Multivariate regression analyses were used to determine the effect of insurance status on LOS, any imaging or procedure, or any AE. RESULTS: Of the 488 patients identified, 220 (45.1%) held governmental insurance while 268 (54.9%) were privately insured. Age was similar between the cohorts (p = 0.616), with the governmental insurance cohort (GI cohort) having a significantly lower proportion of non-Hispanic White patients than the private insurance cohort (PI cohort) (GI: 43.2% vs PI: 72.4%, p < 0.001). While transportation accident was the most common mechanism of injury for both cohorts, assault was significantly greater in the GI cohort (GI: 21.8% vs PI: 3.0%, p < 0.001). A significantly greater proportion of patients in the PI cohort received any imaging (GI: 65.9% vs PI: 75.0%, p = 0.028), while there were no significant differences in procedures performed (p = 0.069) or hospital AEs (p = 0.386) between the cohorts. The median (IQR) LOS (p = 0.186) and discharge disposition (p = 0.302) were similar between the cohorts. On multivariate analysis, with respect to governmental insurance, private insurance was not independently associated with obtaining any imaging (OR 1.38, p = 0.139), undergoing any procedure (OR 1.09, p = 0.721), hospital AEs (OR 1.11, p = 0.709), or LOS (adjusted risk ratio -2.56, p = 0.203). CONCLUSIONS: This study suggests that insurance status may not independently influence healthcare resource utilization and outcomes in adolescent patients presenting with SCIs. Further studies are needed to corroborate these findings.

Draft Genome Sequences of 110 Shiga Toxin-Producing Escherichia coli Isolates Collected from Bovine Manure Samples in the Southern United States.

Shiga toxin-producing Escherichia coli (STEC) bacteria continue to impact the food industry. Environmental sampling of potential sources of contamination is important to aid epidemiologic efforts in tracking foodborne illnesses throughout the United States. Here, the draft genome sequences of 110 STEC isolates from bovine manure collected in Florida and Texas are reported.

CD203c is expressed by human fetal hepatoblasts and distinguishes subsets of hepatoblastoma.

Background & Aims: Hepatocytic cells found during prenatal development have unique features compared to their adult counterparts, and are believed to be the precursors of pediatric hepatoblastoma. The cell-surface phenotype of hepatoblasts and hepatoblastoma cell lines was evaluated to discover new markers of these cells and gain insight into the development of hepatocytic cells and the phenotypes and origins of hepatoblastoma. Methods: Human midgestation livers and four pediatric hepatoblastoma cell lines were screened using flow cytometry. Expression of over 300 antigens was evaluated on hepatoblasts defined by their expression of CD326 (EpCAM) and CD14. Also analyzed were hematopoietic cells, expressing CD45, and liver sinusoidal-endothelial cells (LSECs), expressing CD14 but lacking CD45 expression. Select antigens were further examined by fluorescence immunomicroscopy of fetal liver sections. Antigen expression was also confirmed on cultured cells by both methods. Gene expression analysis by liver cells, 6 hepatoblastoma cell lines, and hepatoblastoma cells was performed. Immunohistochemistry was used to evaluate CD203c, CD326, and cytokeratin-19 expression on three hepatoblastoma tumors. Results: Antibody screening identified many cell surface markers commonly or divergently expressed by hematopoietic cells, LSECs, and hepatoblasts. Thirteen novel markers expressed on fetal hepatoblasts were identified including ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP-3/CD203c), which was found to be expressed by hepatoblasts with widespread expression in the parenchyma of the fetal liver. In culture CD203c+CD326++ cells resembled hepatocytic cells with coexpression of albumin and cytokeratin-19 confirming a hepatoblast phenotype. CD203c expression declined rapidly in culture whereas the loss of CD326 was not as pronounced. CD203c and CD326 were co-expressed on a subset of hepatoblastoma cell lines and hepatoblastomas with an embryonal pattern. Conclusions: CD203c is expressed on hepatoblasts and may play a role in purinergic signaling in the developing liver. Hepatoblastoma cell lines were found to consist of two broad phenotypes consisting of a cholangiocyte-like phenotype that expressed CD203c and CD326 and a hepatocyte-like phenotype with diminished expression of these markers. CD203c was expressed by some hepatoblastoma tumors and may represent a marker of a less differentiated embryonal component.

Sugammadex versus neostigmine for neuromuscular blockade reversal in outpatient surgeries: A randomized controlled trial to evaluate efficacy and associated healthcare cost in an academic center.

Introduction: Neuromuscular blockade is an essential component of the general anesthesia as it allows for a better airway management and optimal surgical conditions. Despite significant reductions in extubation and OR readiness-for-discharge times have been associated with the use of sugammadex, the cost-effectiveness of this drug remains controversial. We aimed to compare the time to reach a train-of-four (TOF) response of ≥0.9 and operating room readiness for discharge in patients who received sugammadex for moderate neuromuscular blockade reversal when compared to neostigmine during outpatient surgeries under general anesthesia. Potential reduction in time for OR discharge readiness as a result of sugammadex use may compensate for the existing cost-gap between sugammadex and neostigmine. Methods: We conducted a single-center, randomized, double arm, open-label, prospective clinical trial involving adult patients undergoing outpatient surgeries under general anesthesia. Eligible subjects were randomized (1:1 ratio) into two groups to receive either sugammadex (Groups S), or neostigmine/glycopyrrolate (Group N) at the time of neuromuscular blockade reversal. The primary outcome was the time to reverse moderate rocuronium-induced neuromuscular blockade (TOF ratio ≥0.9) in both groups. In addition, post-anesthesia care unit (PACU)/hospital length of stay (LOS) and perioperative costs were compared among groups as secondary outcomes. Results: Thirty-seven subjects were included in our statistical analysis (Group S= 18 subjects and Group N= 19 subjects). The median time to reach a TOF ratio ≥0.9 was significantly reduced in Group S when compared to Group N (180 versus 540 seconds; p = 0.0052). PACU and hospital LOS were comparable among groups. Postoperative nausea and vomiting was the main adverse effect reported in Group S (22.2% versus 5.3% in Group N; p = 0.18), while urinary retention (10.5%) and shortness of breath (5.3%) were only experienced by some patients in Group N. Moreover, no statistical differences were found between groups regarding OR/anesthesia, PACU, and total admission costs. Discussion: Sugammadex use was associated with a significantly faster moderate neuromuscular blockade reversal. We found no evidence of increased perioperative costs associated with the use of sugammadex in patients undergoing outpatient surgeries in our academic institution. Clinical trial registration: [https://clinicaltrials.gov/] identifier number [NCT03579589].

Draft Genome Sequences of 278 Salmonella enterica Isolates from Poultry Litter in the Southeastern United States.

Salmonella enterica continues to be a pervasive food safety concern in the poultry industry, contributing to the annual burden of foodborne illnesses in the United States. Poultry litter is a known environmental source for the transmission of Salmonella among broiler flocks. Here, we describe the draft genome sequences of 278 S. enterica isolates collected from poultry litter in Florida.

Survival and inactivation kinetics of Salmonella enterica serovar Typhimurium in irradiated and natural poultry litter microcosms.

The use of poultry litter as a biological soil amendment presents a risk for the preharvest contamination of fresh produce by Salmonella. In order to properly assess this risk, it is important to understand the factors influencing the persistence of Salmonella in poultry litter. This research was performed to investigate the influence of indigenous microflora on the survival of Salmonella Typhimurium in poultry litter. Microcosms of irradiated (sterilized) and natural poultry litter were inoculated with S. Typhimurium, adjusted to pH 8.0, 0.92 water activity (aw), and stored at 30°C for 6 days. S. Typhimurium populations (log CFU g-1) declined in both litter treatments and there were no significant differences (P > 0.05) in recovery between litter treatments on any sampling days (0 to 6). The pH of the natural litter significantly increased (P < 0.05) from 8.42 on day 0 to 9.00 on day 6. By day 6, S. Typhimurium populations in both litter treatments fell below the limit of detection (1 log CFU g-1). The inactivation kinetics of S. Typhimurium in both litter treatments were described by the Weibull model. Under the experimental conditions (pH 8.0, 0.92 aw, 30°C), the presence or absence of poultry litter microflora did not significantly influence the survival of S. Typhimurium. This study demonstrates that the mere presence of poultry litter microflora will not inhibit Salmonella survival. Instead, inhibitory interactions between various microorganisms in litter and Salmonella are likely dependent on more favorable environmental conditions (e.g., aw, pH) for growth and competition.

Factors Impacting Persistence of Phi6 Bacteriophage, an Enveloped Virus Surrogate, on Fomite Surfaces.

The persistence of Phi6 (Φ6) bacteriophage on surfaces commonly encountered in consumer-facing environments was evaluated. Φ6 has been utilized as a surrogate for enveloped viruses, including SARS-CoV-2-the causative agent of COVID-19-due to structural similarities, biosafety level 1 (BSL-1) status, and ease of use. Φ6 persistence on fomites was evaluated by characterizing the impact of the inoculum matrix (artificial saliva, phosphate-buffered saline [PBS], tripartite), inoculum level (low and high), and surface type (nonporous-aluminum, stainless steel, plastic, touchscreen, vinyl; porous-wood). Φ6 was inoculated onto surfaces at low and high inoculum levels for each inoculum matrix and incubated (20.54 ± 0.48°C) for up to 168 h. Φ6 was eluted from the surface and quantified via the double agar overlay assay to determine virus survival over time. For nonporous surfaces inoculated with artificial saliva and PBS, significantly higher D values were observed with high inoculum application according to the 95% confidence intervals. In artificial saliva, D values ranged from 1.00 to 1.35 h at a low inoculum and 4.44 to 7.05 h at a high inoculum across inoculation matrices and surfaces. D values for Φ6, regardless of the inoculum level, were significantly higher in tripartite than in artificial saliva and PBS for nonporous surfaces. In contrast with artificial saliva or PBS, D values in tripartite at low inoculum (D values ranging from 45.8 to 72.8 h) were greater than those at high inoculum (D values ranging from 26.4 to 45.5 h) on nonporous surfaces. This study characterized the impact of the inoculum matrix, inoculum level, and surface type on Φ6 survival on various surfaces relevant to fomite transmission in public settings. IMPORTANCE An important consideration in virus contact transmission is the transfer rate between hands and surfaces, which is driven by several factors, including virus persistence on inanimate surfaces. This research characterized Φ6 persistence on surfaces commonly encountered in public settings based on various factors. The inoculum matrix, which simulates the route of transmission, can impact virus persistence, and three separate matrices were evaluated in this study to determine the impact on Φ6 persistence over time. The number of microorganisms has also been suggested to impact persistence, which was evaluated here to simulate real-world contamination scenarios on six surface types. Results from this study will guide future research utilizing Φ6 or other surrogates for enveloped viruses of public health concern.

Antimicrobial Efficacy of Un-Ionized Ammonia (NH3) against Salmonella Typhimurium in Buffered Solutions with Variable pH, NH3 Concentrations, and Urease-Producing Bacteria.

The presence of Salmonella in poultry litter, when used as a biological soil amendment, presents a risk for the preharvest contamination of fresh produce. Poultry litter is rich in organic nitrogen, and previous studies have suggested that ammonia (NH3) in poultry litter may affect the survival of Salmonella. Salmonella enterica serovar Typhimurium was inoculated into buffer solutions to characterize the pH dependency, minimum antimicrobial concentration, and efficacy of NH3 production. In solutions with 0.4 M total ammonia nitrogen (TAN) at various pH levels (5, 7, 8, and 9), significant inactivation of Salmonella only occurred at pH 9. Salmonella was reduced by ∼8 log CFU/mL within 12 to 18 h at 0.09, 0.18, 0.26, and 0.35 M NH3. The minimum antimicrobial concentration tested was 0.04 M NH3, resulting in an ∼7 log CFU/mL reduction after 24 h. Solutions with urea (1% and 2%) and urease enzymes rapidly produced NH3, which significantly reduced Salmonella within 12 h. The urease-producing bacterium Corynebacterium urealyticum showed no antagonistic effects against Salmonella in solution. Conversely, with 1% urea added, C. urealyticum rapidly produced NH3 in solution and significantly reduced Salmonella within 12 h. Salmonella inactivation data were nonlinear and fitted to Weibull models (Weibull, Weibull with tailing effects, and double Weibull) to describe their inactivation kinetics. These results suggest that high NH3 levels in poultry litter may reduce the risk of contamination in this biological soil amendment. This study will guide future research on the influence of ammonia on the survival and persistence of Salmonella in poultry litter. IMPORTANCE Poultry litter is a widely used biological soil amendment in the production of fresh produce. However, poultry litter may contain human pathogens, such as Salmonella, which introduces the risk of preharvest produce contamination in agricultural fields. Ammonia in poultry litter, produced through bacterial degradation of urea, may be detrimental to the survival of Salmonella; however, these effects are not fully understood. This study utilized aqueous buffer solutions to demonstrate that the antimicrobial efficacy of ammonia against Salmonella is dependent on alkaline pH levels, where increasing concentrations of ammonia led to more rapid inactivation. Inactivation was also demonstrated in the presence of urea and urease or urease-producing Corynebacterium urealyticum. These findings suggest that high levels of ammonia in poultry litter may reduce the risk of contamination in biological soil amendments and will guide further studies on the survival and persistence of Salmonella in poultry litter.

Influence of Free Chlorine and Contact Time on the Reduction of Salmonella Cross-Contamination of Tomatoes in a Model Flume System.

ABSTRACT: The process of washing tomatoes in dump (flume) tanks has been identified as a potential source of cross-contamination. This study's objective was to assess the potential for Salmonella enterica cross-contamination at various inoculation levels in the presence of free chlorine (HOCl) and organic matter. Uninoculated tomatoes were introduced into a laboratory-based model flume containing tomatoes inoculated with a cocktail of five rifampin-resistant S. enterica serovars at 104, 106, or 108 CFU per tomato in water containing 0 or 25 mg/L HOCl and 0 or 300 mg/L chemical oxygen demand (COD). Uninoculated tomatoes exposed to the inoculated tomatoes were removed from the water after 5, 30, 60, and 120 s and placed in bags containing tryptic soy broth supplemented with rifampin and 0.1% sodium thiosulfate. Following incubation, enrichment cultures were plated on tryptic soy agar supplemented with rifampin and xylose lysine deoxycholate agar to determine the presence of Salmonella. HOCl and pH were measured before and after each trial. The HOCl in water containing 300 mg/L COD significantly declined (P ≤ 0.05) by the end of each 120-s trial, most likely due to the increased demand for the oxidant. Higher inoculum levels and lower HOCl concentrations were significant factors (P ≤ 0.05) that contributed to increased cross-contamination. At 25 mg/L HOCl, no Salmonella was recovered under all conditions from uninoculated tomatoes exposed to tomatoes inoculated at 104 CFU per tomato. When the inoculum was increased to 106 and 108 CFU per tomato, cross-contamination was observed, independent of COD levels. The results from this study indicate that the currently required sanitizer concentration (e.g., 100 or 150 mg/L) for flume water may be higher than necessary and warrants reevaluation.

Effects of water activity, ammonia and Corynebacterium urealyticum on the survival of Salmonella Typhimurium in sterile poultry litter.

AIMS: This study examined the effects of water activity (aw ), ammonia and Corynebacterium urealyticum on the survival of Salmonella Typhimurium in sterile poultry litter. METHODS AND RESULTS: Sterile poultry litter inoculated with S. Typhimurium was adjusted to pH 9.0, various aw levels (0.84, 0.92 and 0.96), and total ammonia nitrogen levels were increased either by the addition of ammonium sulphate or C. urealyticum inoculation with 1% urea added. All litter treatments were incubated at 30°C and sampled daily for five days. Similar results were observed at each aw level in both experiments. At 0.84 and 0.92 aw , S. Typhimurium populations in litter fell below 1 log CFU g-1 within 5 days, with no significant differences between the controls and increased ammonia treatments. At 0.96 aw , Salmonella populations treated with increased ammonia levels were significantly lower than control treatments on days 1-5. CONCLUSIONS: This study showed that C. urealyticum can produce ammonia in litter at higher aw levels with sufficient available urea and that the antimicrobial efficacy of ammonia is dependent on high aw (~0.96) in litter. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide insights into the production of ammonia in litter, its antimicrobial efficacy in litter and the importance of aw in this interaction.

Direct characterization of cis-regulatory elements and functional dissection of complex genetic associations using HCR-FlowFISH.

Effective interpretation of genome function and genetic variation requires a shift from epigenetic mapping of cis-regulatory elements (CREs) to characterization of endogenous function. We developed hybridization chain reaction fluorescence in situ hybridization coupled with flow cytometry (HCR-FlowFISH), a broadly applicable approach to characterize CRISPR-perturbed CREs via accurate quantification of native transcripts, alongside CRISPR activity screen analysis (CASA), a hierarchical Bayesian model to quantify CRE activity. Across >325,000 perturbations, we provide evidence that CREs can regulate multiple genes, skip over the nearest gene and display activating and/or silencing effects. At the cholesterol-level-associated FADS locus, we combine endogenous screens with reporter assays to exhaustively characterize multiple genome-wide association signals, functionally nominate causal variants and, importantly, identify their target genes.

Determining Bacterial Load and Water Quality Parameters of Chlorinated Tomato Flume Tanks in Florida Packinghouses.

ABSTRACT: Monitoring and maintenance of water quality in dump tanks or flume systems is crucial to maintaining proper sanitizer levels to prevent pathogen cross-contamination during postharvest washing of tomatoes, but there is limited information on how organic matter influences sanitizer efficacy in the water. The main objective of this study was to monitor water quality in flume tanks and evaluate the efficacy of postharvest washing of tomatoes in commercial packinghouses. Flume tank water samples (n = 3) were collected on an hourly basis from three packinghouses in Florida and analyzed for pH, total dissolved solids (TDS), free chlorine, chemical oxygen demand (COD), oxidation-reduction potential, and turbidity. Additionally, three flume-water samples were collected and tested for total aerobic plate count (APC), total coliforms (TC), and Escherichia coli. Fresh tomatoes (n = 3), both before and after washing, were collected and analyzed for the same bacterial counts. Turbidity, COD, and TDS levels in flume water increased over time in all packinghouses. Correlations observed include COD and turbidity (r = 0.631), turbidity and TDS (r = 0.810), and oxidation-reduction potential and chlorine (r = 0.660). APC for water samples had an average range of 0.0 to 4.7 log CFU/mL and TC average range of 0.0 to 4.7 log CFU/mL. All water samples were negative for E. coli. The average APC for pre- and postflume tomatoes from the three packinghouses was 6.0 log CFU per tomato and ranged from 2.2 to 7.4 log CFU per tomato. The average TC count was <1.5 and 7.0 log CFU per tomato for pre- and postwash tomatoes, respectively. There was no significant effect (P > 0.05) of postharvest washing on the microbiological qualities of tomatoes. Water quality in flume tanks deteriorated over time in all packinghouses during a typical operational day of 4 to 8 h.

A bioinspired and chemically defined alternative to dimethyl sulfoxide for the cryopreservation of human hematopoietic stem cells.

The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.

Andersen-Tawil Syndrome with High Risk of Sudden Cardiac Death in Four Mexican Patients. Cardiac and Extra-Cardiac Phenotypes

ABSTRACT Background: Andersen-Tawil syndrome (ATS) is a cardiac channelopathy that is inherited in an autosomal dominant way, and it is characterized by a triad of periodic paralysis, ventricular arrhythmias, and includes some dysmorphic features with incomplete penetrance and variable expression that result in a challenging diagnosis. Objective: The objective of the study was to describe the cardiac and extra-cardiac phenotype in a cohort of patients with ATS at risk of sudden cardiac death (SCD) to improve its early clinical identification. Methods: In an observational, transversal study, with a deviant case sampling, four female patients with ATS at high risk of SCD were included in the study. They carried the heterozygous pathogenic variants c.407C>T [p.Ser136Phe], c.652C>T [p.Arg218Trp] (n=2), and c.431G>C [p.Gly144Ala] in the KCNJ2 gene. Patients were evaluated by a cardiologist, a clinical geneticist, and a physiatrist. Results: One patient had the classical facial phenotype and the other three had subtle manifestations. The group of patients presented a diverse set of clinical data such as: triangular face, broad forehead, broadening of medial eyebrows, auricular pits, low-set ears, eyelid ptosis, thin lips, mandibular hypoplasia, and diverse types of dental alterations, single transverse palmar crease, camptodactyly, and syndactyly. Long-exercise test showed a decrement in the percentage amplitude up to 44%, classifying patients in IV or V types according to Fournier’s scale. Conclusions: Extra-cardiac manifestations were a common finding in this series of ATS type1 at high risk of SCD. Its recognition could help the clinician in the early identification of patients with ATS, especially for the cardiologist since they are commonly referred only for evaluation of ventricular arrhythmias.