Assembly of a multivalent aptamer for efficient inhibition of thermostable direct hemolysin toxicity induced by Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) is a key virulence factor of Vibrio parahaemolyticus, capable of causing seafood-mediated outbreaks of gastroenteritis, posing a threat to the aquatic environment and global public health. In the present study, we explored a multivalent aptamer-mediated inhibition strategy to mitigate TDH toxicity. Based on the characteristic structure of TDH, a stable multivalent aptamer, Ap3-5, was rationally designed by truncation, key fragment evolution, and end fixation. Ap3-5 exhibited strong affinity (Kd=39.24 nM), and thermal (Tm=57.6 °C) and enzymatic stability. In silico studies also revealed that Ap3-5 occupied more active sites of TDH and covered its central pore, indicating its potential as a blocking agent for inhibiting TDH toxicity. In the hemolysis assay, Ap3-5 significantly suppressed the hemolytic effect of TDH. A cellular study revealed a substantial (∼80 %) reduction in TDH cytotoxicity. Supporting these findings, in vivo trials confirmed the inhibitory action of Ap3-5 on both the acute and intestinal toxicity of TDH. Overall, benefiting from the strong binding affinity, high stability, and multisite occupation of the multivalent aptamer with TDH, Ap3-5 displayed robust potential against TDH toxicity by inhibiting membrane pore formation, providing a new approach for alleviating bacterial infections.

Tdh3 and Rom2 are functional modulators of a conserved condensate-resident RNA-binding protein, Scd6 in Saccharomyces cerevisiae.

RGG-motif proteins play a crucial role in determining mRNA fate. Suppressor of clathrin deficiency 6 (Scd6) is a conserved RGG-motif containing RNP condensate-resident, translation repressor, and decapping activator protein in Saccharomyces cerevisiae. Identifying protein factors that can modulate Scd6 function is critical to understanding the regulation of mRNA fate by Scd6. In this study, using an approach that combined mRNA tethering assay with flow cytometry, we screened 50 genes for their role in modulating the translation repression activity of Scd6. We identified eight conserved modulators with human homologs. Of these, we further characterised in detail guanine nucleotide exchange factor (GEF) Rom2 (Rho1 multicopy suppressor) and glycolytic enzyme Tdh3 (Triose phosphate dehydrogenase 3), which, respectively, impede and promote translation repression activity of Scd6. Our study reveals that Rom2 negatively regulates the arginine methylation of Scd6 and antagonises its localisation to P-bodies. Tdh3, on the other hand, promotes Scd6 interaction with Hmt1, thereby promoting the arginine methylation of Scd6 and enhanced eIF4G1 interaction, which is known to promote its repression activity. Identifying these novel modulators provides exciting new insights into the role of a metabolic enzyme of the glycolytic pathway and guanine nucleotide exchange factor implicated in the cell wall integrity pathway in regulating Scd6 function and, thereby, cytoplasmic mRNA fate.

Bases neurobiológicas de la epilepsia neonatal y sus comorbilidades / Neurobiological basis of neonatal epilepsy and its co morbilities

Resumen La aparición de convulsiones es frecuente durante el periodo neonatal debido a las características de inma durez funcional del cerebro es este periodo. La aparición de estas convulsiones puede llevar a un diagnóstico de epilepsia neonatal, que suele estar asociado a alteracio nes estructurales del cerebro durante el neurodesarrollo. Aproximadamente el 50% de las personas con epilepsia activa padecen al menos un trastorno médico comórbi do, y esto hace que cambie la evolución de la epilepsia. La presencia de trastornos neurológicos que preceden a la aparición de la epilepsia indica que alteraciones es tructurales y/o funcionales del cerebro subyacentes pue den ser causa de la predisposición a padecer epilepsia y de los procesos comórbidos de manera independiente. En esta revisión describimos los procesos cerebrales estructurales y funcionales que subyacen a la aparición de epilepsia neonatal y sus comorbilidades.

Abstract The occurrence of seizures is frequent during the neonatal period due to the functional immaturity of the brain.The presence of these seizures may lead to a diagnosis of neonatal epilepsy, which is usually as sociated with structural alterations of the brain during neurodevelopment. Approximately 50% of people with active epilepsy have at least one comorbid medical di sorder, and the existence of a comorbid process changes the course of the epilepsy. The presence of neurologic disorders preceding the onset of epilepsy indicates that underlying neurobiological alterations may indepen dently cause the predisposition to epilepsy and comor bid processes. In this review we describe the structural and functional brain processes underlying the onset of neonatal epilepsy and its comorbidities.

Utility of somatosensory- and motor-evoked potential change thresholds in surgical treatment for thoracic spinal stenosis based on different pathologies.

BACKGROUND CONTEXT: Thoracic spinal stenosis (TSS) is secondary to different pathologies that differ in clinical characteristics and surgical outcomes. PURPOSE: This study aimed to determine the optimal warning thresholds for combined somatosensory-evoked potentials (SSEP) and motor-evoked potentials (MEP) for predicting postoperative neurological deterioration in surgical treatment for TSS based on different pathologies. Additionally, we explored the correlation between SSEP/MEP monitoring and postoperative spinal neurological function. STUDY SETTING: Retrospective study. PATIENT SAMPLE: Two hundred five patients. OUTCOME MEASURES: We obtained perioperative modified Japanese Orthopedic Association (mJOA) scores to assess spinal neurological function. METHODS: The data collected in this study included demographic data, intraoperative neurophysiological monitoring (IONM) signals, and perioperative neurological function assessments. To determine the optimal IONM warning threshold, a receiver operating characteristic (ROC) curve was used. Additionally, Pearson correlation analysis was conducted to determine the correlation between IONM signals and clinical neurological conditions. RESULTS: A total of 205 consecutive patients were eligible. Forty-one patients had thoracic disc herniation (TDH), 14 had ossification of the posterior longitudinal ligament (OPLL), 124 had ossification of the ligamentum flavum (OLF), and 26 had OPLL+OLF. The mean mJOA scores before surgery and 3 months after surgery were 7.0 and 7.9, respectively, resulting in a mean mJOA recovery rate (RR) of 23.1%. The average postoperative mJOA RRs for patients with TDH, OPLL, OLF, and OPLL+OLF were 24.8%, 10.4%, 26.8%, and 11.2%, respectively. Patients with OPLL+OLF exhibited a more stringent threshold for IONM changes. This included a lower amplitude cutoff value (a decrease of 49.0% in the SSEP amplitude and 57.5% in the MEP amplitude for short-term prediction) and a shorter duration of waveform change (19.5 minutes for SSEP and 22.5 minutes for MEP for short-term prediction). On the other hand, patients with TDH had more lenient IONM warning criteria (a decrease of 49.0% in SSEP amplitude and 77.5% in MEP amplitude for short-term prediction; durations of change of 25.5 minutes for SSEP and 32.5 minutes for MEP). However, OPLL patients or OLF patients had moderate and similar IONM warning thresholds. Additionally, there was a stronger correlation between the SSEP amplitude variability ratio and the JOA RR in OPLL+OLF patients, while the correlation was stronger between the MEP amplitude variability ratio and the JOA RR for the other three TSS pathologies. CONCLUSIONS: Optimal IONM change criteria for prediction vary depending on different TSS pathologies. The optimal monitoring strategy for prediction varies depending on TSS pathologies.

Laparoscopic Repair Following a Delayed Presentation of Traumatic Diaphragmatic Hernia: A Case Report.

Traumatic diaphragmatic hernia is a rare condition that occurs after trauma, and some patients have a delayed presentation. A laparoscopic approach is rarely used to repair traumatic diaphragmatic hernias. We encountered a case of asymptomatic diaphragmatic hernia diagnosed after a comprehensive medical examination. A 71-year-old woman was diagnosed with a delayed presentation of traumatic diaphragmatic hernia with prolapse of the greater omentum owing to a traffic injury 20 years ago. Surgery was performed laparoscopically using three ports, and intraoperative respiratory management was performed using a double-lumen tube. The 2.5-cm-diameter hernial orifice was sutured under contralateral one-lung ventilation after the greater omentum was returned to the abdominal cavity. The patient's postoperative course was uneventful, and she was discharged on the third day. Intraoperative strategies such as respiratory management and the laparoscopic approach play a crucial role in ensuring favorable postoperative outcomes. The last follow-up was at six months post-operation, and the patient was doing well.

[Neurobiological basis of neonatal epilepsy and its comorbilities]. / Bases neurobiológicas de la epilepsia neonatal y sus comorbilidades.

The occurrence of seizures is frequent during the neonatal period due to the functional immaturity of the brain.The presence of these seizures may lead to a diagnosis of neonatal epilepsy, which is usually associated with structural alterations of the brain during neurodevelopment. Approximately 50% of people with active epilepsy have at least one comorbid medical disorder, and the existence of a comorbid process changes the course of the epilepsy. The presence of neurologic disorders preceding the onset of epilepsy indicates that underlying neurobiological alterations may independently cause the predisposition to epilepsy and comorbid processes. In this review we describe the structural and functional brain processes underlying the onset of neonatal epilepsy and its comorbidities.

La aparición de convulsiones es frecuente durante el periodo neonatal debido a las características de inmadurez funcional del cerebro es este periodo. La aparición de estas convulsiones puede llevar a un diagnóstico de epilepsia neonatal, que suele estar asociado a alteraciones estructurales del cerebro durante el neurodesarrollo. Aproximadamente el 50% de las personas con epilepsia activa padecen al menos un trastorno médico comórbido, y esto hace que cambie la evolución de la epilepsia. La presencia de trastornos neurológicos que preceden a la aparición de la epilepsia indica que alteraciones estructurales y/o funcionales del cerebro subyacentes pueden ser causa de la predisposición a padecer epilepsia y de los procesos comórbidos de manera independiente. En esta revisión describimos los procesos cerebrales estructurales y funcionales que subyacen a la aparición de epilepsia neonatal y sus comorbilidades.

Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus.

Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 °C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity.

Fitness and transcriptomic analysis of pathogenic Vibrio parahaemolyticus in seawater at different shellfish harvesting temperatures.

IMPORTANCE: Given the involvement of Vibrio parahaemolyticus (Vp) in a wide range of seafood outbreaks, a systematical characterization of Vp fitness and transcriptomic changes at temperatures of critical importance for seafood production and storage is needed. In this study, one of each virulent Vp strain (tdh+ and trh+) was tested. While no difference in survival behavior of the two virulent strains was observed at 10°C, the tdh+ strain had a faster growth rate than the trh+ strain at 30°C. Transcriptomic analysis showed that a significantly higher number of genes were upregulated at 30°C than at 10°C. The majority of differentially expressed genes of Vp at 30°C were annotated to functional categories supporting cellular growth. At 10°C, the downregulation of the biofilm formation and histidine metabolism indicates that the current practice of storing seafood at low temperatures not only protects seafood quality but also ensures seafood safety.

Incidence of Traumatic Diaphragmatic Injury: Results From a Retrospective Cohort Study in a Level I Trauma Center in Riyadh.

Introduction Traumatic diaphragmatic injuries (TDIs) are uncommon and the incidence of TDI is difficult to estimate because of the variation in reporting missed or late detected diaphragmatic injuries. Therefore, our study's aim was to investigate the prevalence of traumatic diaphragmatic injury in the thoracoabdominal trauma, discuss the etiological factors, diagnostic investigations, and outcomes in TDIs, and evaluate predictors of mortality in patients who were diagnosed at King Abdulaziz Medical City in Riyadh, Saudi Arabia. Materials and methods This observational retrospective cohort study was conducted at King Abdulaziz Medical City (KAMC), a tertiary hospital in Riyadh, Saudi Arabia. The study included all adult patients aged 18 years or older diagnosed with traumatic diaphragmatic injuries between the years 2016 and 2020. The BESTCare electronic system was used to get the patient's medical records and extract the data. Pearson χ2 test was used for categorical variables, and an independent t-test was used for continuous variables to investigate the association between predictors and outcomes.  Results A total of eight patients were involved in this study. The mean age of the patients was 49 years old. Males outnumbered females by 75%. Patients admitted with blunt injuries were greater by 75% compared to penetrating injuries by 25%. The left side of the diaphragm was the most common site of injury. The total number of patients who were admitted to the ICU was five, four of whom had blunt trauma and one had a penetrating injury. Conclusion The demographic data of the patients included in this study corresponded to that in the literature. Although not reaching a statistically significant level, ICU admissions and mortality were mostly associated with blunt injuries. Larger multi-center studies are required to further investigate the incidence of traumatic diaphragmatic injuries (TDI).

Laparoscopic Repair of Acute Traumatic Diaphragmatic Hernia: A Case Report.

Traumatic diaphragmatic hernia (TDH) is a rare condition resulting from blunt or penetrating thoracoabdominal trauma and is characterized by the protrusion of abdominal organs into the thoracic cavity through a ruptured diaphragm. Due to its diverse clinical presentations, TDH often faces diagnostic challenges. Accurate diagnosis relies on imaging studies and surgical exploration, with surgical intervention being the primary treatment approach. This case presentation highlights a young patient who presented to Saint George Hospital following a blunt thoracoabdominal injury. The patient experienced unexplained dyspnea upon admission, and imaging revealed herniated bowels in the left hemithorax. Laparoscopic exploration confirmed a left hemi-diaphragmatic tear, with the transverse colon, omentum, most of the small bowel, and stomach herniating into the left hemithorax. The patient underwent laparoscopic repair, involving the reduction of the herniated organs into the peritoneal cavity and tension-free primary closure with gastropexy without the use of mesh for reinforcement. The patient's postoperative course was uneventful, and complete recovery was achieved. This case report provides insights into the diagnosis and management of TDH, highlighting the importance of prompt recognition and appropriate surgical intervention in achieving successful outcomes.

Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus.

Vibrio parahaemolyticus is usually found in seafood and causes acute gastroenteritis in humans. Therefore, a detection method of pathogenic V. parahaemolyticus is necessary. Multiplex PCR combined with lateral flow dipstick (LFD) assay was developed to detect pathogenic V. parahaemolyticus. Biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes were used for multiplex PCR amplification. The condition of the method was optimized and evaluated by agarose gel electrophoresis and universal lateral flow dipstick. The specificity assay was evaluated using strains belonging to seven foodborne pathogen species. The sensitivity of the method was also evaluated using DNA in the concentration range of 0.39-100 ng/reaction. The artificial spiking experiment was performed using 10 g of shrimp samples with an enrichment time of 0, 4, and 8 h with 101, 102, and 103 CFU of V. parahaemolyticus. The developed multiplex PCR-LFD assay showed no non-specific amplification with a limit of the detection of 0.78 ng DNA/reaction visualized by agarose gel electrophoresis and 0.39 ng DNA with LFD assay. The artificial spiking experiment demonstrated that this method could detect pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples following a 4 h of enrichment. Multiplex PCR-LFD assay was therefore established for detecting pathogenic V. parahaemolyticus with high sensitivity and specificity and might be a useful tool to develop a detection kit used in the food safety sector.

Systematic Approaches to Study Eclipsed Targeting of Proteins Uncover a New Family of Mitochondrial Proteins.

Dual localization or dual targeting refers to the phenomenon by which identical, or almost identical, proteins are localized to two (or more) separate compartments of the cell. From previous work in the field, we had estimated that a third of the mitochondrial proteome is dual-targeted to extra-mitochondrial locations and suggested that this abundant dual targeting presents an evolutionary advantage. Here, we set out to study how many additional proteins whose main activity is outside mitochondria are also localized, albeit at low levels, to mitochondria (eclipsed). To do this, we employed two complementary approaches utilizing the α-complementation assay in yeast to uncover the extent of such an eclipsed distribution: one systematic and unbiased and the other based on mitochondrial targeting signal (MTS) predictions. Using these approaches, we suggest 280 new eclipsed distributed protein candidates. Interestingly, these proteins are enriched for distinctive properties compared to their exclusively mitochondrial-targeted counterparts. We focus on one unexpected eclipsed protein family of the Triose-phosphate DeHydrogenases (TDH) and prove that, indeed, their eclipsed distribution in mitochondria is important for mitochondrial activity. Our work provides a paradigm of deliberate eclipsed mitochondrial localization, targeting and function, and should expand our understanding of mitochondrial function in health and disease.

Evaluation of Alternative Colony Hybridization Methods for Pathogenic Vibrios.

Vibrios, such as Vibrio parahaemolyticus, are naturally occurring halophilic bacteria that are a major cause of foodborne illness. Because of their autochthonous nature, managing vibrio levels in marine and estuarine environments is impossible. Instead, it is crucial to reliably enumerate their abundance to minimize human exposure. One method of achieving this is the direct plating/colony hybridization (DP/CH) method, which has been used to efficiently quantify pathogenic vibrios in oysters and other seafood products. Although successful, the method relies on proprietary resources. We examined alternative approaches, assessed the influence of the reagent suppliers' source on enumeration accuracy, and made experimental adjustments that maximized efficiency, sensitivity, and specificity. We report here that in-house conjugation via Cell Mosaic is a viable alternative to the previously available sole-source distributor of the alkaline phosphatase-conjugated probes used to enumerate vibrios in oysters. We also report that milk was a viable alternative as a blocking reagent, pH must be eight, an orbital shaker was a viable alternative to a water bath, and narrow polypropylene containers were a viable alternative to Whirl-Pak bags. These modifications will be crucial to scientists enumerating vibrios and other pathogens in food products.

Effect of sunlight and salinity on the survival of pathogenic and non-pathogenic strains of Vibrio parahaemolyticus in water microcosms.

The effect of sunlight and salinities (10, 20, 39, and 60 psu) on the survival of Vibrio parahaemolyticus strains carrying either (thermostable direct hemolysin) tdh, the (thermostable related hemolysin) trh, and both or none of them were studied in water microcosms stabilized at 20°C using plate count agar and acridine orange direct viable count. All V. parahaemolyticus strains exposed to sunlight rapidly lose their culturability and evolve into a viable but non-culturable state (VBNC). However, the tdh positive strains remain more culturable than the non-virulent or trh positive strain but statically insignificant. At tested salinities, the survival time was higher at 10, 20, and 60 psu compared with that observed in seawater (39 psu). In seawater under dark condition, Vibrio strains remain culturable for up to 200 days with a significant difference between strains (p < 0.05). Furthermore, the non-pathogenic strain survives longer than the virulent ones. At different salinities, a better adaptation is observed at 10 and 20 psu compared with 39 and 60 psu. Resuscitations essays performed on VBNC bacteria in a nutrient broth at 20°C and 37°C does not show any revivification. PRACTITIONER POINTS: Effect of sunlight and salinities on the survival of V. parahaemolyticus in the marine environment. Resuscitation essay performed on viable but no cultivable bacteria. Microscope motility examines show that all strains exposed to sunlight remain motile after the loss of cultivability.

Trh positive strain of Vibrio parahaemolyticus induce immunity by modulating MAPK pathway: A molecular pathogenic insight in immune-related gene regulation.

Vibrio parahaemolyticus is a zoonotic bacterium that causes infections in shellfish, fish and higher vertebrates as well as in humans. The Tdh and Trh positive strains of V. parahaemolyticus are generally considered as major virulent strains. The pathogenic mechanisms of Trh positive strain of V. parahaemolyticus are poorly understood. Therefore, in the present study Indian Major Carp, Labeo rohita was intraperitoneally challenged with a Trh positive strain of V. parahaemolyticus below lethal dose 50 (LD50) to understand the innate immune response. A significant upregulation in the respiratory burst activity, myeloperoxidase activity and lysozyme activity of serum was observed in the challenged fishes. However, the serum alpha (α) 2-macro globulin activity and antiprotease activity remained unaltered in the infected fish. The relative expression study of some immune-related genes showed that after the experimental challenge the expression of immune-related genes viz., Toll-like receptor (TLR), Nucleotide-binding oligomerization domain (NOD), Interleukin-1ß (IL-ß), Interleukin-6 (IL-6), Tumor necrosis factor α (TNFα), Inducible nitric oxide synthase (iNOS), Complement factor 3a (C3a) and Heat shock proteins 70 (Hsp70) was upregulated during infection. Furthermore, overexpression of nuclear factor kappa light chain enhancer of activated B cells (NF-κß), Myeloid differentiation primary response 88 (MyD88), Mitogen-activated protein kinases (MAPK) and cysteine-aspartic proteases (Casp 1) was also observed after post-infection which clearly indicated that Trh positive V. parahaemolyticus activates MAPK pathway. The present study strengthens the understanding of molecular pathogenesis and provides insights on gene regulation during infection with Trh positive V. parahaemolyticus.

Virulence factor genes and comparative pathogenicity study of tdh, trh and tlh positive Vibrio parahaemolyticus strains isolated from Whiteleg shrimp, Litopenaeus vannamei (Boone, 1931) in India.

Vibrio parahaemolyticus is a gram-negative halophilic bacterium responsible for gastrointestinal infection in human and vibriosis in aquatic animals. The thermostable direct hemolysin (tdh), tdh-related hemolysin (trh) and thermolabile hemolysin (tlh) positive strains of V. parahaemolyticus were identified from brackishwater aquaculture farms of West Bengal and Andhra Pradesh, India. Moreover, the presence of other virulent genes like vcrD1, vopD, vp1680 under type three secretion system 1 (T3SS1) and vcrD2 vopD2, vopB2, vopC2 under type three secretion system 2 (T3SS2) were detected in tdh positive strain of V. parahaemolyticus. Furthermore, the study revealed that the tdh and trh positive isolates were resistant to ß-lactam antibiotics and were able to lyse more than 95% of human Red Blood Cells (RBCs). In addition, both the isolates showed high cytotoxicity in Human Embryonic Kidney (HEK) cell line compared to tlh positive strain. Additionally, intraperitoneal and oral administration of tdh and trh positive strain of V. parahaemolyticus in Indian Major Carp, Labeo rohita caused 100% mortality at the level of 2.0 × 108 CFU ml-1 and 1.6 × 108 CFU ml-1, respectively. In contrast, only 10% mortality was observed in the case of tlh positive strain at the level of 2.5× 108 CFU ml-1. The histopathological changes like infiltration of blood cells and degenerated hepatic tissue in the liver of L. rohita were observed after the experimental challenge. The changes like degeneration of glomeruli, necrosis of renal tubules and Bowman's capsule were observed in the kidney section. Ragged, irregular shaped villi and necrosis of the villus were observed in the intestinal lumen. Overall, the study demonstrates that isolated V. parahaemolyticus is a potent aquatic microbial pathogen. Additionally, as V. parahaemolyticus is also a human pathogen and might pose a threat to the human population, proper management strategies are required to prevent the possible occurrence of disease.

Mutational sources of trans-regulatory variation affecting gene expression in Saccharomyces cerevisiae.

Heritable variation in a gene's expression arises from mutations impacting cis- and trans-acting components of its regulatory network. Here, we investigate how trans-regulatory mutations are distributed within the genome and within a gene regulatory network by identifying and characterizing 69 mutations with trans-regulatory effects on expression of the same focal gene in Saccharomyces cerevisiae. Relative to 1766 mutations without effects on expression of this focal gene, we found that these trans-regulatory mutations were enriched in coding sequences of transcription factors previously predicted to regulate expression of the focal gene. However, over 90% of the trans-regulatory mutations identified mapped to other types of genes involved in diverse biological processes including chromatin state, metabolism, and signal transduction. These data show how genetic changes in diverse types of genes can impact a gene's expression in trans, revealing properties of trans-regulatory mutations that provide the raw material for trans-regulatory variation segregating within natural populations.

Antibiotic resistance and determination of resistant genes among cockle (Anadara granosa) isolates of Vibrio alginolyticus.

A total of 24 strains of Vibrio alginolyticus were isolated from cockles (Anadara granosa) and identified for VibA and gyrB genes. All V. alginolyticus isolates were then tested against nine different antibiotics. In this study, the highest percentage of antibiotic resistance was obtained against penicillin (37.50%), followed by ampicillin, vancomycin (12.50%) and erythromycin (8.33%). All of V. alginolyticus isolates were susceptible against streptomycin, kanamycin, tetracycline, chloramphenicol and sulfamethoxazole. Polymerase chain reaction (PCR) assay has confirmed the presence of four antibiotic resistance genes of penicillin (pbp2a), ampicillin (blaOXA), erythromycin (ermB) and vancomycin (vanB). Out of 24 V. alginolyticus isolates, 2 isolates possessed the tdh-related hemolysin (trh) (strains VA15 and VA16) and none for the thermostable direct hemolysin (tdh) gene. Both strains of the tdh-related hemolysin (trh) were susceptible to all antibiotics tested. The multiple antibiotic resistance (MAR) index ranging between 0.2 and 0.3 with 5 antibiograms (A1-A5) was observed. Combination of enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and antibiotic resistance indicated 18 genome types which showed genetic heterogeneity of those V. alginolyticus isolates. The results demonstrated the presence of V. alginolyticus strain found in cockles can be a potential risk to consumers and can contribute to the deterioration of human health in the study area. Thus, it is essential for local authority to provide the preventive measures in ensuring the cockles are safe for consumption.

Longitudinal Study of Total and Pathogenic Vibrio parahaemolyticus (tdh+ and/or trh+) in Two Natural Extraction Areas of Mytilus chilensis in Southern Chile.

Vibrio parahaemolyticus is the leading cause of seafood-associated bacterial gastroenteritis worldwide. Although different studies have focused on its pattern of variation over time, knowledge about the environmental factors driving the dynamics of this pathogen, within the Chilean territory, is still lacking. This study determined the prevalence of total and pathogenic V. parahaemolyticus strains (tdh and/or trh genes) in mussels (Mytilus chilensis) collected from two natural growing areas between 2017 and 2018, using selective agar and PCR analysis. V. parahaemolyticus was detected in 45.6% (93/204) of pooled samples from the Valdivia River Estuary. The pathogenic strains carrying the tdh and/or trh gene were detected in 11.8% (24/204): tdh in 9.8% (20/204), trh in 0.5% (1/204), and 1.5% (3/204) presented both genes. In Reloncaví Fjord, V. parahaemolyticus was detected in 14.4% (30/209) of the samples, pathogenic V. parahaemolyticus carrying the trh gene was detected in 0.5% (1/209) of the samples, while the tdh gene was not detected in the samples from this area. The total count of mauve-purple colonies typical of V. parahaemolyticus on CHROMagar was positively associated by multivariate analysis with area, water temperature, and salinity. Similarly, V. parahaemolyticus detection rates by PCR had a positive correlation with the area and water temperature. The chances of detecting total V. parahaemolyticus in the Valdivia River Estuary are significantly higher than in the Reloncaví Fjord, but inversely, during spring-summer months, the interaction factor between the area and temperature indicated that the chances of detecting V. parahaemolyticus are higher in the Reloncaví Fjord. Interestingly, this period coincides with the season when commercial and natural-growing shellfish are harvested. On the other hand, pathogenic V. parahaemolyticus tdh+ was significantly correlated with an increase of water temperature. These environmental parameters could be used to trigger a warning on potential hazard, which would influence human health and economic losses in aquaculture systems.

The identification and determination of toxin genes of Vibrio strains causing hemorrhagic disease on red drum (Sciaenops ocellatus) using PCR.

Data were collected from 30 strains of Vibrio and sampled on different organs (brain, hemorrhagic site and digestive tract) of Sciaenops ocellatus infection. The results showed that the nucleotide sequences 16S rRNA region are highly similar to those of V. alginolyticus, V. azureus, V. fluvialis, V. natriengens and V. orientalis, which were published on Genbank and other, ranging from 98.05 to 100%. The digestive tract has the most common Vibrio strains (V. alginolyticus [16] V. azureus [7] and V. fluvialis). Thereout, 25 of 30 strains of Vibrio contained 1 to 3 toxin genes, except V. parahaemolyticus. Six parameters were used to measure the DNA polymorphism of 33 homologous DNA sequences in this Vibrio bacteria population. The results indicated that number of separate polymorphic sites (S), total number of mutant sites (Eta), number of haplotype (h), haplotype diversity (Hd), average number of nucleotide differences (k), nucleotide diversity (Pi) were 98 (S), 103 (Eta), 9 (h), 0.887 ± 0.032 (Hd), 25.789 (k) and 17.980 × 10-3 ± 0.003 (Pi), respectively (P < 0,05). The G + C content above 1434 sites positions of nucleotide sequences accounted for 0.542. The phylogenetic tree showed that these strains are divided into six groups. As observed, the appearance of isolated Vibrio on 3 organs of fish (S. ocellatus) hemorrhagic are V. azureus (27,67%), V. alginolyticus (50%), V. orientalis (6,67%) and V. fluvialis (16,67%). Through this result, we found that the diversity of Vibrio species that appeared on the red drum was used in the 16S rRNA region and the presence of toxin genes in these Vibrio species.