RFX4 is an intrinsic factor for neuronal differentiation through induction of proneural genes POU3F2 and NEUROD1.

Proneural genes play a crucial role in neuronal differentiation. However, our understanding of the regulatory mechanisms governing proneural genes during neuronal differentiation remains limited. RFX4, identified as a candidate regulator of proneural genes, has been reported to be associated with the development of neuropsychiatric disorders. To uncover the regulatory relationship, we utilized a combination of multi-omics data, including ATAC-seq, ChIP-seq, Hi-C, and RNA-seq, to identify RFX4 as an upstream regulator of proneural genes. We further validated the role of RFX4 using an in vitro model of neuronal differentiation with RFX4 knock-in and a CRISPR-Cas9 knock-out system. As a result, we found that RFX4 directly interacts with the promoters of POU3F2 and NEUROD1. Transcriptomic analysis revealed a set of genes associated with neuronal development, which are highly implicated in the development of neuropsychiatric disorders, including schizophrenia. Notably, ectopic expression of RFX4 can drive human embryonic stem cells toward a neuronal fate. Our results strongly indicate that RFX4 serves as a direct upstream regulator of proneural genes, a role that is essential for normal neuronal development. Impairments in RFX4 function could potentially be related to the development of various neuropsychiatric disorders. However, understanding the precise mechanisms by which the RFX4 gene influences the onset of neuropsychiatric disorders requires further investigation through human genetic studies.

Distinct effect of exercise modes on mood-related behavior in mice.

Exercise can afford several benefits to combat mood disorders in both rodents and humans. Engagement in various physical activities upregulates levels of neurotrophic factors in several brain regions and improves mental health. However, the type of exercise that regulates mood and the underlying mechanisms in the brain remain elusive. Herein, we performed two distinct types of exercise and RNA sequencing analyses to investigate the effect of exercise on mood-related behaviors and explain the distinct patterns of gene expression. Specifically, resistance exercise exhibited reduced immobility time in the forced swim test when compared with both no exercise and treadmill exercise (in the aerobic training [AT] group). Interestingly, anxiety-like behaviors in the open field and nest-building tests were ameliorated in the AT group when compared with those in the control group; however, this was not observed in the RT group. To elucidate the mechanism underlying these different behavioral changes caused by distinct exercise types, we examined the shift in the gene expression pattern in the hippocampus, a brain region that plays a critical role in regulating mood. We discovered that 38 and 40 genes were altered in the AT and RT groups, respectively, compared with the control group. Both exercises regulated 16 common genes. Compared with the control group, mitogen-activated protein kinase (MAPK) was enriched in the AT group and phosphatidylinositol-3-kinase (PI3K)/AKT and neurotrophin signaling pathways were enriched in the RT group, as determined by bioinformatics pathway analysis. PCR results revealed that Cebpß expression was increased in AT group, and Dcx expression was upregulated in both groups. Our findings indicate that different exercise types may exert substantially distinct effects on mood-like behaviors. Accordingly, appropriate types of exercise can be undertaken based on the mood disorder to be regulated.

Semantic variant primary progressive aphasia with a pathogenic variant p.Asp40Gly in the ANXA11 gene.

We report a patient with right-predominant semantic variant primary progressive aphsia linked with p.Asp40Gly variant of ANXA11, which is the first description of frontotemporal dementia without clinical and electrophysiological evidences of amyotrophic lateral sclerosis associated with a known pathogenic variant of ANXA11.

Sample collection for pre-transfusion crossmatching: Benefits of using an electronic identification system.

BACKGROUND: Transfusion of ABO blood group-mismatched blood or administration to the wrong recipient may result in fatal adverse events. To prevent these types of errors, various strategies have been employed. Recently, we developed a novel sample collection workflow for the pre-transfusion crossmatching test and patient recognition. This study aimed to analyse the usage of the new workflow and improvements in outcomes. METHODS: We analysed the number of crossmatching and wrong-patient errors among the blood transfusion cases during 3 years of data collection (from August 2018 to July 2021). From May 2021 to July 2021, the new workflow was implemented. Outcomes were calculated according to the department type, patient age and processing time. The sample processing time was defined as the time from placing the order to lab arrival. RESULTS: The new workflow utilisation increased from 50.7% to 80.3% and wrong-patient errors decreased annually. The new workflow was used for more adults (3001/3680 samples, 81.5%) than paediatric cases (345/522 samples, 65.5%; p < 0.001) and in general wards than in the emergency room or intensive care unit. The sample processing time differed according to ward type and timing of the request (day: 28.80, 2.43-3889.43 min, night: 3.36, 2.72-1671.47 min; p < 0.001). CONCLUSION: Wrong-patient errors were reduced without increasing sample-processing time after introducing the new workflow which included using an electronic identification system. The time needed for the blood processing differed according to the ward type, patient age, and timing of the request. Patient safety can be promoted by managing these factors and using an electronic identification system.

Evaluation of conductivity-based osmolality measurement in urine using the Sysmex UF5000.

BACKGROUND: Automated flow cytometry-based urine analyzer is increasingly being used to identify and enumerate cells and particles in urine specimens. It measures electrical conductivity which could be transformed to osmolality. Using this machine, all urine specimens could be screened for osmolality without requiring a separate dedicated device. We evaluated the performance of the new instrument, the UF-5000 (Sysmex Corporation), in the measurement of urine osmolality. METHODS: The precision of urine osmolality measurement by the UF-5000 was evaluated for 20 days and 4 times a day for 2 concentrations. The linearity and detection capability were evaluated according to the Clinical and Laboratory Standards Institute guidelines. For comparison, 270 random urine specimens from patients were tested simultaneously using the UF5000 and the OsmoPro micro-osmometer (Advanced instruments). RESULTS: The laboratory-based coefficient variations were less than 5%. Urine osmolality using the UF-5000 has a verified linear range (y = 1.097x + 16.91, R2  = .997). Within the comparison analysis, the mean difference was not large (-7.72%) but each differences were largely dispersed with 95% limits of agreement (LoA) from -70.5 to 55.06%, and the mean absolute difference -28.3 mOsm/kg with 95% LoA from -295.13 to 238.45 mOsm/kg. Cohen's kappa value was 0.54 (95% CI, 0.45-0.63). CONCLUSIONS: The UF-5000 measured conductivity and generated an acceptable quantitative analysis of urine osmolality. When compared with the results of the freezing point depression method used by the OsmoPro, a percentage of the measured urine osmolality by the UF-5000 was outside the allowable limit.

Comparison of Serologic Response of Hospitalized COVID-19 Patients Using 8 Immunoassays.

BACKGROUND: In Korea, there were issues regarding the use of immunoassays for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies to detect infection. So, we compared antibody results of eight kinds of commercial immunoassays using clinical remnant specimens. METHODS: We compared the results of several immunoassay kits tested on 40 serum samples from 15 confirmed patients and 86 remnant serum samples from clinical laboratory. Eight kinds of IVD kits-four enzyme-linked immunosorbent assay, two lateral flow rapid immunochromatographic assays, and two chemiluminescent immunoassays with one RUO kit were tested. RESULTS: Among 40 serum samples from 15 coronavirus disease 2019 (COVID-19) patients, 35 yielded at least one positive result for detecting antibodies in the combined assessment. There were inconsistent results in 12 (28%) samples by single immunoassay. Forty samples collected in 2019 before the first COVID-19 Korean case showed negative results except for one equivocal result. CONCLUSION: The discrepant results obtained with different immunoassay kits in this study show that serological assessment of SARS-CoV-2 by a single immunoassay requires caution not only in detecting infection but also in assessing immunologic status.

Intragenic CpG islands play important roles in bivalent chromatin assembly of developmental genes.

CpG, 5'-C-phosphate-G-3', islands (CGIs) have long been known for their association with enhancers, silencers, and promoters, and for their epigenetic signatures. They are maintained in embryonic stem cells (ESCs) in a poised but inactive state via the formation of bivalent chromatin containing both active and repressive marks. CGIs also occur within coding sequences, where their functional role has remained obscure. Intragenic CGIs (iCGIs) are largely absent from housekeeping genes, but they are found in all genes associated with organ development and cell lineage control. In this paper, we investigated the epigenetic status of iCGIs and found that they too reside in bivalent chromatin in ESCs. Cell type-specific DNA methylation of iCGIs in differentiated cells was linked to the loss of both the H3K4me3 and H3K27me3 marks, and disruption of physical interaction with promoter regions, resulting in transcriptional activation of key regulators of differentiation such as PAXs, HOXs, and WNTs. The differential epigenetic modification of iCGIs appears to be mediated by cell type-specific transcription factors distinct from those bound by promoter, and these transcription factors may be involved in the hypermethylation of iCGIs upon cell differentiation. iCGIs thus play a key role in the cell type-specific regulation of transcription.

PACT increases mammalian embryonic neural stem cell properties by facilitating activation of the notch signaling pathway.

The protein activator of protein kinase R (PKR) (PACT) is known to play important roles in PKR regulation and microRNA biogenesis. Based on the observation that PACT is specifically expressed in the ventricular zone (VZ) at the mid-neurogenic period, we examine the role of PACT in this embryonic neural stem cell niche. Here, we provide the first evidence that PACT increases neurosphere formation, as well as expression of Notch target genes and the neural stem cell marker Sox2 in primary neural stem cells in vitro. Consistently, introduction of PACT into the mouse embryonic brain in utero increased the fraction of cells localizing to the VZ. We also show that the PACT-enhanced stemness of neural stem cells is PKR-independent. At the molecular level, PACT was revealed to physically interact with C promoter binding factor 1 (CBF1) and dramatically strengthen the association between CBF1 and Notch intracellular domain (NICD), which indicates stabilization of the Notch transcriptional coactivation complex responsible for Notch target gene expression. Taken together, our study indicates that PACT is a novel transcriptional coactivator of the Notch pathway playing a pivotal role during mammalian brain development.

Risk factors of rescue bismuth quadruple therapy failure for Helicobacter pylori eradication.

BACKGROUND AND AIM: Failure of bismuth quadruple therapy for Helicobacter pylori eradication is frequently observed. To increase the eradication rate, comprehensive analyses need to be performed regarding risk factors of bismuth quadruple therapy failure based on complete standard culture and antimicrobial susceptibility testing results. METHODS: Patients with history of failed first therapy who had H. pylori colonies isolated from culture and successful minimum inhibitory concentration (MIC) test were enrolled. Esomeprazole, bismuth, metronidazole, and tetracycline (quadruple) therapies for 7 or 14 days were given. Eradication rate, treatment compliance, adverse events, and risk factors for the failure of bismuth quadruple therapy were analyzed. RESULTS: A total 54 patients were enrolled. Overall eradication rate in the present study was 88.8%. The eradication rate for cases with metronidazole resistance such as MIC 8-16 µg/mL or 16-32 µg/mL was 92.8% (13/14). For cases with high level metronidazole resistance (MIC > 32 µg/mL), the eradication rate was only 60% (6/10). Multivariate analysis regarding compliance, treatment duration, age > 60, three kinds of metronidazole MICs, tetracycline MIC > 4 µg/mL, adverse events and any other parameters, "metronidazole resistance, high level (MIC > 32 µg/mL)" was the only independent risk factor for eradication failure (P = 0.007). CONCLUSION: For cases with metronidazole resistance at MIC > 32 µg/mL, rescue therapy other than bismuth-containing quadruple therapy is needed.

Prevalence, incidence, and mortality of nontuberculous mycobacterial infection in Korea: a nationwide population-based study.

BACKGROUND: Epidemiologic characteristics of nontuberculous mycobacterial (NTM) disease remain largely unknown. The objective of this study was to evaluate incidence, prevalence, and mortality of NTM infection in a large nationwide population-based cohort in Korea. METHODS: Data of the National Health Insurance Service database, an extensive health-related database including most Korean residents, were used. Adults with a primary diagnosis of NTM as determined by International Classification of Disease-Tenth Revision coding (A31) were identified between 2003 and 2016. Incidence, prevalence, and mortality of NTM infection were analyzed. RESULTS: A total of 46,194 individuals had a primary diagnosis of NTM infection. Their mean age was 55.8 years. Of these subjects, 61.1% were females. Annual age-adjusted incidence and prevalence of NTM infection tended to increase rapidly from 2003 to 2016. Age-adjusted incidence and prevalence was 17.9 and 33.3 per 100,000 population in 2016. The incidence and prevalence were higher in females and the elderly. The 5-year mortality rate in the population with NTM infection was 17.8%. The standardized mortality ratio of patients with NTM infection to the general population was 2.16 (95% confidence interval: 2.10 to 2.22). CONCLUSIONS: This large population-based study showed that the incidence and prevalence of NTM infection in Korea increased rapidly from 2003 to 2016. They were higher in women and the elderly. The mortality rate in the population with NTM infection was higher than that in the general population.

rdxA, frxA, and efflux pump in metronidazole-resistant Helicobacter pylori: Their relation to clinical outcomes.

BACKGROUND AND AIM: rdxA and frxA mutations and enhancement of efflux pump have been suggested as the cause of metronidazole resistance in Helicobacter pylori. This study was performed to investigate the resistance mechanisms related to clinical eradication outcome, and to examine direct involvement of hefA in metronidazole-resistant isolates with intact rdxA and frxA. METHODS: A total of 53 H. pylori-positive patients who were treated with metronidazole-containing sequential or quadruple therapy from 2011 to 2015 were enrolled. The metronidazole susceptibility of H. pylori isolates was examined by agar dilution test. Mutations in rdxA and frxA, were analyzed with DNA sequencing, and impact of hefA on metronidazole resistance was examined with quantitative real-time reverse transcription polymerase chain reaction, knockout and genetic complementation test for hefA. RESULTS: Seven mutation types of rdxA and/or frxA were found in H. pylori isolated from non-eradicated subjects. rdxA mutation was associated with eradication failure (P = 0.002), and nonsense mutation in rdxA reduced eradication efficacy (P = 0.009). hefA expression was significantly higher in resistant isolates (P < 0.001), especially in rdxA(-)frxA(-) as compared to rdxA(+)frxA(+) (P = 0.027). Resistant isolates with no mutation in rdxA and frxA became susceptible after hefA knockout. Genetic complementation for hefA recovered metronidazole resistance in all of three hefA knockout mutants. CONCLUSIONS: These results suggest that rdxA mutations play a critical role in metronidazole resistance as well as the outcomes of eradication therapy. In addition, hefA seems to be directly involved in metronidazole resistance, which explains the resistance in clinical isolates with intact rdxA and frxA.

Specific mutations of penicillin-binding protein 1A in 77 clinically acquired amoxicillin-resistant Helicobacter pylori strains in comparison with 77 amoxicillin-susceptible strains.

BACKGROUND: Amoxicillin (Amx) is one of the most important antibiotics for eradication of Helicobacter pylori (H. pylori). Main determinants of genetically stable Amx resistance are mutations in the C-terminus of penicillin-binding protein 1A (pbp1A). However, contribution of individual mutation remains unclear. METHODS: 77 Amx-resistant (AmxR ) and 77 Amx-susceptible (AmxS ) H. pylori strains were isolated from gastric tissues, and DNA sequencing was performed to compare C-terminus sequences of pbp1A gene between AmxR and AmxS strains. Natural transformation of these mutated genes into amoxicillin-susceptible strains was performed. RESULTS: Among many mutations in pbp1A, D479E (OR: 37.4, 95% CI: 5.53-252.49, P < .001), and T593 mutation (OR: 32.0, 95% CI: 4.04-252.86, P < .001) independently contributed to Amx resistance in H. pylori strains. In the transformation experiment, T593 mutations were identified in their transformants showing Amx resistance. However, PCR product of D479E was not inserted into recipient (ATCC 43504) resulting in transformation failure. CONCLUSION: Amx resistance is associated with various substitutions in pbp1A and T593 mutation contributes to Amx resistance of H. pylori.

MAL and TMEM220 are novel DNA methylation markers in human gastric cancer.

CONTEXT: Gastric cancer (GC) is the fourth most common cause of cancer-related deaths worldwide. OBJECTIVE: To determine the mRNA-expression of the MAL, TMEM220, MMP28, IL-19 and HOPX genes and analyse the methylation statuses of MAL and TMEM220. MATERIALS AND METHODS: Gene-expression levels were analysed in 10 GC cell lines and 30 matched pairs of GC and normal mucosa (NM) gastric tissue specimens in real-time reverse-transcriptase polymerase chain reactions. Gene methylation was evaluated by bisulphite sequencing. Detailed gene-methylation patterns were confirmed by pyrosequencing analysis. RESULTS: MAL, TMEM220, MMP28 and IL-19 were significantly down-regulated in GC cell lines and GC tissues compared to NM tissues. MAL and TMEM220 were highly methylated in GC tissues, and methylation inversely correlated with expression. MAL and TMEM220 expression were restored by treatment with 5-aza-2'-deoxycytidine. MAL and TMEM220 were specifically methylated and were down-regulated in human GC. DISCUSSION AND CONCLUSION: These loci may serve as novel methylation markers for patients with GC.

Increasing use of therapeutic apheresis as a liver-saving modality.

INTRODUCTION: Therapeutic plasma exchange (TPE) is used for temporary support of liver function in patients presenting with early graft dysfunction after liver transplantation (LT) or liver surgery. We analyzed the effect of therapeutic apheresis on patients with liver disease. METHODS: Between January 2011 and August 2016, 93 apheresis procedures were performed for 26 patients at our institution. Anti-ABO isoagglutination immunoglobulin (Ig) M titer was checked using a type A and type B 3% red blood cell (RBC) suspension in saline with two-fold serial dilutions of patient serum. Anti-ABO isoagglutination IgG titer was checked by a type A and B 0.8% RBC suspension using a low-ionic strength/Coombs card. RESULTS: ABO-incompatible (ABOi) LT was the most common (n=10, 38.5%) indication for apheresis; early graft dysfunction after LT (n=8, 30.7%) was the second most common. Median initial IgM and IgG anti-ABO titers for ABOi LT recipients were 1:16 (range, 1:8-1:128) and 1:48 (range, 1:8-1:2048). We performed preoperative TPE in 10 recipients (median number of sessions, 1.5; range, 1-11). Among patients with early graft dysfunction, those who underwent living donor LT had better survival (4/4; 100%) than those who underwent nonliving donor LT (0/3; 0%). Patients who underwent living donor LT first and then additional LT also survived after three TPE sessions. CONCLUSION: Therapeutic apheresis is associated with a good survival rate and is essential for liver support in patients with early graft dysfunction after LT or posthepatectomy liver failure and during preparation for ABOi LT.

HBx induces hypomethylation of distal intragenic CpG islands required for active expression of developmental regulators.

Epigenetic alterations caused by viral oncoproteins are strong initiation factors for cancer development, but their mechanisms are largely unknown. To identify the epigenetic effects of viral hepatitis B virus X (HBx) that lead to hepatocellular carcinoma (HCC), we profiled the DNA methylomes of normal and HBx transgenic mouse liver. Intriguingly, severe hypomethylation of intragenic CpG islands (CGIs) was observed in HBx liver before the full development of HCC. Normally, these CGIs were highly methylated (mCGIs) by the DNMT3L complex and marked with epigenetic signatures associated with active expression, such as H3K36me3. Hypomethylation of mCGI was caused by the downregulation of Dnmt3L and Dnmt3a due to HBx bound to their promoters, along with HDAC1. These events lead to the downregulation of many developmental regulators that could facilitate tumorigenesis. Here we provide an intriguing epigenetic regulation mediated by mCGI that is required for cell differentiation and describe a previously unidentified epigenetic role for HBx in promoting HCC development.

Effectiveness of Bilateral Arm Training for Improving Extremity Function and Activities of Daily Living Performance in Hemiplegic Patients.

BACKGROUND: Bilateral movement therapy, which encourages simultaneous use of the limbs on both the affected and nonaffected sides, is known to help in motor function recovery in hemiplegic patients. However, studies on the effectiveness of bilateral arm training for improving upper limb function and activities of daily living (ADL) performance in hemiplegic stroke patients are lacking. The present study investigated the effectiveness of bilateral arm training for improving upper limb function and ADL performance in hemiplegic stroke patients. METHODS: The study included 30 hemiplegic stroke patients. The patients were randomly divided into an experimental group (n = 15) and a control group (n = 15). All patients received a uniform general occupational therapy session lasting 30 minutes 5 times a week for 8 weeks. The experimental group received an additional session of bilateral arm training lasting 30 minutes, and the control group received an additional session of general occupational therapy lasting 30 minutes. The Fugl-Meyer assessment (FMA), Box and Block Test (BBT), and modified Barthel index (MBI) were used for evaluation. RESULTS: In both the experimental and control groups, the FMA, BBT, and MBI scores were significantly higher after the intervention than before the intervention (P <.05). The changes in the FMA, BBT, and MBI scores were greater in the experimental group than in the control group (P <.05). CONCLUSIONS: Bilateral arm training along with general occupational therapy might be more effective than occupational therapy alone for improving upper limb function and ADL performance in hemiplegic stroke patients.

ChARM: Discovery of combinatorial chromatin modification patterns in hepatitis B virus X-transformed mouse liver cancer using association rule mining.

BACKGROUND: Various chromatin modifications, identified in large-scale epigenomic analyses, are associated with distinct phenotypes of different cells and disease phases. To improve our understanding of these variations, many computational methods have been developed to discover novel sites and cell-specific chromatin modifications. Despite the availability of existing methods, there is still room for further improvement when they are applied to resolve the histone code hypothesis. Hence, we aim to investigate the development of a computational method to provide new insights into de novo combinatorial pattern discovery of chromatin modifications to characterize epigenetic variations in distinct phenotypes of different cells. RESULTS: We report a new computational approach, ChARM (Combinatorial Chromatin Modification Patterns using Association Rule Mining), that can be employed for the discovery of de novo combinatorial patterns of differential chromatin modifications. We used ChARM to analyse chromatin modification data from the livers of normal (non-cancerous) mice and hepatitis B virus X (HBx)-transgenic mice with hepatocellular carcinoma, and discovered 2,409 association rules representing combinatorial chromatin modification patterns. Among these, the combination of three histone modifications, a loss of H3K4Me3 and gains of H3K27Me3 and H3K36Me3, was the most striking pattern associated with the cancer. This pattern was enriched in functional elements of the mouse genome such as promoters, coding exons and 5'UTR with high CpG content, and CpG islands. It also showed strong correlations with polymerase activity at promoters and DNA methylation levels at gene bodies. We found that 30 % of the genes associated with the pattern were differentially expressed in the HBx compared to the normal, and 78.9 % of these genes were down-regulated. The significant canonical pathways (Wnt/ß-catenin, cAMP, Ras, and Notch signalling) that were enriched in the pattern could account for the pathogenesis of HBx. CONCLUSIONS: ChARM, an unsupervised method for discovering combinatorial chromatin modification patterns, can identify histone modifications that occur globally. ChARM provides a scalable framework that can easily be applied to find various levels of combination patterns, which should reflect a range of globally common to locally rare chromatin modifications.

Pellet feed adsorbed with the recombinant Lactococcus lactis BFE920 expressing SiMA antigen induced strong recall vaccine effects against Streptococcus iniae infection in olive flounder (Paralichthys olivaceus).

The aim of this study was to develop a fish feed vaccine that provides effective disease prevention and convenient application. A lactic acid bacterium (LAB), Lactococcus lactis BFE920, was modified to express the SiMA antigen, a membrane protein of Streptococcus iniae. The antigen was engineered to be expressed under the nisin promoter, which is induced by nisin produced naturally by the host LAB. Various sizes (40 ± 3.5 g, 80 ± 2.1 g, and 221 ± 2.4 g) of olive flounder (Paralichthys olivaceus) were vaccinated by feeding the extruded pellet feed, onto which the SiMA-expressing L. lactis BFE920 (1.0 × 10(7) CFU/g) was adsorbed. Vaccine-treated feed was administered twice a day for 1 week, and priming and boosting were performed with a 1-week interval in between. The vaccinated fish had significantly elevated levels of antigen-specific serum antibodies and T cell marker mRNAs: CD4-1, CD4-2, and CD8a. In addition, the feed vaccine significantly induced T cell effector functions, such as the production of IFN-γ and activation of the transcription factor that induces its expression, T-bet. When the flounder were challenged by intraperitoneal infection and bath immersion with S. iniae, the vaccinated fish showed 84% and 82% relative percent survival (RPS), respectively. Furthermore, similar protective effects were confirmed even 3 months after vaccination in a field study (n = 4800), indicating that this feed vaccine elicited prolonged duration of immunopotency. In addition, the vaccinated flounder gained 21% more weight and required 16% less feed to gain a unit of body weight compared to the control group. The data clearly demonstrate that the L. lactis BFE920-SiMA feed vaccine has strong protective effects, induces prolonged vaccine efficacy, and has probiotic effects. In addition, this LAB-based fish feed vaccine can be easily used to target many different pathogens of diverse fish species.