Stable Marker Research Articles

Stable bull fertility protein markers in seminal plasma

Bull fertility is an important trait in breeding as the semen of one bull can, potentially, be used to perform thousands of inseminations. The high number of inseminations needed to obtain reliable measures from Non-Return Rates to oestrus creates difficulties in assessing fertility accurately. Improving molecular knowledge of seminal properties may provide ways to facilitate selection of bulls with good semen quality. In this study, liquid chromatography mass spectrometry (LC-MS/MS) was used to analyze the protein content from the seminal plasma of 20 bulls with Non-Return Rates between 35 and 60%, sampled across three seasons. Overall, 1343 proteins were identified and proteins with consistent correlation to fertility across multiple seasons found. From these, nine protein groups had a significant Pearson correlation (p < 0.1) with fertility in all three seasons and 34 protein groups had a similar correlation in at least two seasons. Among notable proteins showing a high and consistent correlation across seasons were Osteopontin, a lipase (LIPA) and N-acetylglucosamine-1phosphotransferase subunit gamma. Three proteins were combined in a multiple linear regression to predict fertility (r = 0.81). These sets of proteins represent potential markers, which could be used by the breeding industry to phenotype bull fertility. SignificanceThe ability of bull spermatozoa to fertilize oocytes is crucial for breeding efficiency. However, the reliability of this trait from field measures is relatively low and the prediction of fertility given by conventional methods to evaluate sperm quality is currently not very accurate. In this work, we identify sets of proteins in bull seminal plasma from repeated samples collected at different times of the year that correlate to fertility in a consistent way. We combined these individual proteins to build a molecular signature predictive of fertility. This study provides an overview of proteins linked to fertility in seminal plasma, thereby increasing knowledge of the bull seminal plasma proteome. Protein signatures from the latter, potentially related to fertility, may be of use to predict fertility for individual bulls.

Association analysis of physiological traits in spring barley (Hordeum vulgare L.) under water-deficit conditions.

In the present study, 148 commercial barley cultivars were assessed by 14 AFLP primer combinations and 32 SSRs primer pairs. Population structure, linkage disequilibrium, and genomic regions associated with physiological traits under drought stress were investigated. The phenotypic results showed a high level of diversity between studied cultivars. The studied barley cultivars were divided into two subgroups. Linkage disequilibrium analysis revealed that r 2 values among all possible marker pairs have an average value of 0.0178. The mixed linear model procedure showed that totally, 207 loci had a significant association with investigated traits. 120 QTLs out of 207 were detected for traits under normal conditions, and 90 QTLs were detected for traits under drought stress conditions. Identified QTLs after validation and transferring to SCAR markers in the case of AFLPs can be used to develop MAS strategies for barley breeding programs. Some common markers were identified for a particular trait or some traits across normal and drought stress conditions. These markers show low interaction with environmental conditions (stable markers); therefore, selection by them for a trait under normal conditions will improve the trait value under stress conditions, too.

Evidence for seasonal changes in population structure of Reed Buntings (Emberiza schoeniclus) in the Lake Neusiedl Region: a stable-isotope approach

Migrating species and populations are affected by conditions existing across large geographical scales. The degree to which populations mix at stop-over sites and wintering grounds is variable and can involve important fitness consequences. Thus, the link between breeding and wintering grounds and seasonal shifts in population structure are important to understand population demographics, local adaption and diversification patterns. This work aims to infer migration patterns and population connectivity in the Reed Bunting (Emberiza schoeniclus) community of a Ramsar-designated wetland, Lake Neusiedl, Seewinkel, Austria. Stable isotope markers (hydrogen, δ2H, carbon, δ13C) in feathers were analyzed to assign individuals to a specific breeding or natal area. Based on the geographic assignment, birds were compared with respect to their ecomorphological traits and their habitat selection (large reed bed versus more open habitat at shore of an alkaline lake) during winter to assess potential ecological differences and possibly associated morphological divergence. Stable isotope data revealed that the local Reed Bunting community undergoes seasonal changes in population structure. Three different populations that vary in their δ2H signature and to some extent in morphological characteristics as well as in δ13C values were identified. Indications for differential migration related to age and/or sex were additionally found, since males and females as well as first-year birds and adults showed remarkable differences in their traits and feather isotope values. δ2H values diverged significantly between these groups in both age and sex classes and birds with highest feather δ2H values were considered to represent the local breeding population at Lake Neusiedl and possibly also Reed Buntings from close surroundings. Moreover, this group had the highest δ13C values in first-year individuals as well as in adults. In both sex classes, the assignment of Reed Buntings to different populations was ultimately possible due to differences in bill shape with residents being characterized by more massive bills than individuals belonging to populations visiting the study area only during migration or for wintering. During winter, beak shape and feather δ2H and δ13C values differed significantly between birds caught in the reed stand of Lake Neusiedl and those captured near the salt pan, hence, indicating differential habitat selection among co-occurring populations during winter. These findings suggest that morphological attributes, such as bill shape and size as well as linked adaptations to different food sources play a decisive role for niche segregation among wintering populations. The results provide evidence that different populations mix during migration and during winter at Lake Neusiedl and the extent to which they are specialized to a specific habitat varied.

Induction and early identification of tetraploid black locust by hypocotyl in vitro

Tetraploid black locust (Robinia pseudoacacia L.) has advantages over diploid varieties in growth and stress resistance. Previous studies had shown that tetraploid black locust can be induced only by treating shoot tips of the germinating seeds with colchicine. In order to establish a simpler and more efficient tetraploid induction method in black locust, we induced tetraploid plants by treating the hypocotyls from in vitro–grown seedlings with different concentrations of colchicine, and then provided a method for tetraploid identification based on stomatal size and density in this study. The highest induction efficiency was 53.33% by treating hypocotyl explants with 70 mg·L−1 colchicine for 2 d. Tetraploid black locust was significantly different from diploid in morphological characteristics. Leaf area, leaf thickness, chlorophyll content, stomatal length, and stomatal width were greater in tetraploid plants than in diploid plants, whereas the opposite was true for leaf aspect ratio, stomatal index, and stomatal density. The differences in stomatal size and density between diploid and tetraploid were nearly two times; therefore, size and density of stomata can be used as a stable and distinct marker to rapidly screen tetraploids. The results of this study suggested that the hypocotyl was an ideal material to induce tetraploid black locust in vitro and confirmed that stomatal size and density could be an effective and reliable marker to identify ploidy level in black locust.

Expression of CD304/neuropilin-1 in adult b-cell lymphoblastic leukemia/lymphoma and its utility for the measurable residual disease assessment.

Many new markers are being evaluated to increase the sensitivity and applicability of multicolor flow cytometry (MFC)-based measurable residual disease (MRD) monitoring. However, most of the studies are limited to childhood B-cell lymphoblastic leukemia/lymphoma (B-ALL), and reports in adult B-ALL are extremely scarce and limited to small cohorts. We studied the expression of CD304/neuropilin-1 in a large cohort of adult B-ALL patients and evaluated its practical utility in MFC-based MRD analysis. CD304 was studied in blasts from adult B-ALL patients and normal precursor B cells (NPBC) from non-B-ALL bone marrow samples using MFC. CD304 expression intensity and pattern were studied with normalized-mean fluorescent intensity (nMFI) and coefficient of variation of immunofluorescence (CVIF), respectively. MFC-based MRD was performed at end of induction (EOI; day-35), end of consolidation (EOC; day 78-80), and subsequent follow-up (SFU) time points. CD304 was positive in 120/214(56.07%) and was significantly associated with BCR-ABL1 fusion (P=.001). EOI-MRD and EOC-MRD were positive in 129/214(60.3%) and 50/81(61.72%), respectively. CD304 was positive in a significant percentage of EOI (48%, 62/129) and EOC (52%, 26/50) MRD-positive B-ALL samples. Its expression was retained, lost, and gained in 73.7%, 26.3%, and 11.3% of EOI-MRD and 85.7%, 14.3%, and none of EOC-MRD samples, respectively. Low-level MRD (<0.01%) was detectable in 34 of all (EOI+EOC+SFU=189) MRD-positive samples, and CD304 was found useful in 50% of these samples. CD304 is commonly expressed in adult B-ALL and clearly distinguish B-ALL blasts from normal precursor B cells. It is a stable MRD marker and distinctly useful in the detection of MFC-based MRD monitoring, especially in high-sensitivity MRD assay.

Identification of genomic regions at seedling related traits in response to aluminium toxicity using a new high-density genetic map in rice (Oryza sativa L.)

Aluminium toxicity is adversely affecting the rice growth and production in acidic soils of developing countries. Rice is popular and stable crop which meets food requirements of about 50% world population. A set of 122 rice backcross recombinant inbreed lines (BRILs) derived from a cross between Japonica (cv.02428) and Indica (Changhui/891) was grown in nutrient solution. High density linkage map generated by using 3075 highly stable SNP markers. Relative root length (RRL) was used as standard criteria for aluminium tolerance. A total of 23 QTLs were detected in the current study for six traits across all experimental conditions. Regarding RRL, three QTLs qCRL-6, qSRL-6, and qRRL-6 were found stable and common QTLs across all environments. Out of these three QTLs, qRRL-6 with higher heritability estimates (H2 = 96.59%) was novel QTL with a positive additive effect. In addition, shoot length (SL), shoot fresh weight (SFW), root fresh weight (RFW), shoot dry weight (SDW) and root dry weight (RDW) were used and identified QTLs, qSSL-11, qRRFW-1, and qRRDW-1 and qRRDW-8 which were newly reported QTLs. Three pleiotropic QTLs qRRFW-1, qRSDW-1, and qRRDW-1 were also reported and these QTLs would facilitate to improve Al tolerance by MAS selection and QTLs pyramiding.

Advances allowing feasible pyrG gene editing by a CRISPR-Cas9 system for the edible mushroom Pleurotus eryngii

For decades, the edible mushroom Pleurotus eryngii (P. eryngii) has been cultivated as important raw materials for food and pharmaceutical industries in most of Asian countries, especially in China. Unfortunately, the generation and improvement of new cultivars are very difficult since there are many barriers which have not been solved thoroughly by gene editing tools, even though the CRISPR-Cas9 technique has been widely applied in other species. In this study, we identified the point-mutated variant of the endogenous sdhB gene (cbxr) as a more stable selection marker than hygromycin B resistance gene (hph) in P. eryngii. Furthermore, using a codon-optimized Cas9, a predicted native U6 promoter-guided sgRNA, as well as an optimized protoplast transformation system, a highly efficient pyrG gene editing system was established in P. eryngii, that incorporated varied insertions and deletions (indels) by non-homologous end joining (NHEJ) and homology-directed repair (HDR). Findings for a successful targeted gene editing strategy in the edible mushroom P. eryngii may open a new chapter for the improvement of edible mushroom cultivars.

Значимость персональных клинико-диагностических показателей при оценке побочного радиационного воздействия для больных раком щитовидной железы, получающих радиойодтерапию

The radioiodine therapy is the most well-known branch of radionuclide therapy. The therapy composed of thyroidectomy and iodine-131 radiopharmaceutical is the “gold standard” for treat-ment of differentiated thyroid cancer patients (DTC). Given metastases in lymph nodes such therapy is absolutely exclusive treatment mode. At the same time, despite the targeted effect of radioiodine on pathological foci a side internal exposure of healthy organs and tissues is noted in the course of radioiodine therapy. Therefore, a reliable assessment of the side exposure is nec-essary taking into account the individual characteristics of patients considering that the side dose varies significantly from patient to patient. For this reason, the identification of significance of personal clinic-diagnostic factors determined the side whole body exposure is the vital and im-portant task. In this aspect, the cytogenetic examination of DTC patients by means of the analysis of chromosomal aberrations in peripheral blood lymphocytes is the goal of essential importance. The certain types of chromosomal aberrations are specific radiation markers that unequivocally indicate radiation exposure. Using the frequency of radiation markers the side absorbed dose can be reliably estimated. In the present work, the statistical analysis of the significance of vari-ous clinic-diagnostic factors in relation to the induction of radiation markers in the blood lympho-cytes was performed. For that end the results of the cytogenetic examination of the group com-prised of 38 DTC patients have been used. The examined patients underwent a course of radio-iodine therapy in the department of radionuclide therapy of the A.F. Tsyb MRRC, Obninsk. The slide preparation and cytogenetic analysis were carried out in the laboratory of radiation cytoge-netic of the same Center. The performed analysis of the correlation matrix with respect to the dependence of the increased frequency of stable and unstable markers resulted from a one-time course of radioiodine therapy showed no meaningfulness at the level R&gt;0.3 for all the studied factors. For the absolute value of the frequency of radiation markers, both before and after radioiodine therapy, the meaningful relationship was found at the level R&gt;0.7 for unsta-ble and at the level R&gt;0.8 for stable markers with a total administered activity of radioiodine dur-ing all the previous courses of radioiodine therapy.

Psychological Factors in Fibromyalgia and Chronic Fatigue Syndrome: Implications in Counseling

The condition of Chronic Fatigue Syndrome (CFS) includes fatigue and exhaustibility, muscle pain, muscle weakness, sore throat, fever, headache, impaired memory, concentration difficulties and sleep disorder. Whereas Fibromyalgia (FM) include symptoms such as aches, pain, stiffness and powerlessness in the muscles, fatigue, exhaustibility, headache, swelling, numbness, bowel problems, and sleeping difficulties Sometimes, fibromyalgia patients report anxiety, tension, insomnia, and depressive mood, while some patients reported psychiatric disorders such as major depressive and anxiety disorders. Fibromyalgia (FM) often categorized, as a ‘functional somatic syndrome’, or sometimes, ‘somatization disorder’. Both CFS and FM share the symptomatology to a large extent. Patients with fibromyalgia also meet the criteria for chronic fatigue syndrome whereas individuals with chronic fatigue syndrome also manifest concurrent fibromyalgia. In absence of any biomedical test or a stable physiological marker, researchers also conceptualize chronic fatigue syndrome as a psychiatric disorder, or manifestation of a psychiatric condition such as somatization disorder. The present paper is a review of the studies conducted in the area to understand the nature, clinical picture and causal factors for these conditions. It is also aimed to provide the information about the underlying mechanism of CFS and FM to counsellors to incorporate this body of knowledge while counselling with the similar kind of problems.

Non-pegylated Liposomal Doxorubicin as Palliative Chemotherapy in pre-Treated Advanced Pancreatic Cancer: A Retrospective Analysis of Twenty-Eight Patients.

Background: Pancreatic cancer carries a devastating prognosis and is the fourth leading cause for cancer-related death in the United States and most European countries. Although one-third of patients receive a palliative third line therapy, the benefit of systemic therapy beyond second-line remains unclear. A plethora of clinical trials investigating novel drugs have failed over the past years. Due to the lack of established treatment regimens beyond second line, we offered nonpegylated liposomal doxorubicin, well known in other tumor entities, to pretreated pancreatic cancer patients requesting systemic therapy. Material and Methods: In this retrospective analysis, 28 patients with pancreatic carcinoma treated with nonpegylated liposomal doxorubicin (Myocet®) between 2012 and 2018 at our department were included. Results: For the majority of patients (n = 18, 64%), nonpeglyted liposomal doxorubicin was offered as a third-line therapy. Five patients received it as second line, four patients as fourth line, and one patient as fifth line of therapy. Half of the patients received at least a therapy cycle. The objective response rate to treatment was 7.1%. One patient had a period of radiologically confirmed stable disease with stable tumor markers. Another patient experienced partial remission. Conclusion: According to our findings the benefit of nonpegylated liposomal doxorubicin in pancreatic cancer beyond second line is limited.

Copeptin levels are more elevated and associated with cardiovascular events in the unity of chronic ischemic heart and chronic kidney diseases

Objective: Copeptin comprises the C-terminal part of the Arginine vasopressin (AVP) precursor, is co-secreted with AVP and is a stable surrogate marker for AVP release, making it much easier to measure. Elevated levels of serum copeptin in both cardiovascular and renal diseases were known but the amount and predictive value of copeptin in the unity of these diseases is unknown.&#x0D; Methods: A study composed equally divided five groups, total of 150 adult patients. Group I and group II composed study groups which included the patients of chronic ischemic heart (CHID) and chronic kidney diseases (CKD) respectively. Control groups such as group III, IV and V included only patients of CHID or CKD. Copeptin, hs-CRP and pro-BNP levels were analyzed in all patients and their relationship with adverse outcomes were evaluated.&#x0D; Results: Serum levels of copeptin, hs-CRP and pro-BNP were higher in patients of study groups than patients of control groups and it was found statistically meaningful (p

Diagnostic Utility of C4d by Direct Immunofluorescence in Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease that commonly affects elderly patients. Direct immunofluorescence (DIF) for immunoglobulin G (IgG) and C3c on frozen skin biopsies is the gold standard for the diagnosis of BP. In a minority of cases, IgG and/or C3c are found negative, and in these situations, there is a need for a more stable diagnostic marker of BP. C4d is biologically inactive, but has a long half-life, rendering it a long-lived marker for antibody-mediated complement activation. Previous studies already demonstrated that C4d was diagnostically useful in formalin-fixed paraffin-embedded skin biopsies of patients with BP. We hypothesized that C4d detected by DIF could also be a promising diagnostic marker for BP, particularly in IgG and/or C3c DIF-negative cases. In this single-center retrospective study, 69 cases of BP were analyzed for linear deposition of C4d; of the 69 cases, n = 26 were IgG+/C3c-, n = 10 IgG+/C3c+, and n = 33 IgG-/C3c-. Results were compared with n = 39 negative controls. Seven of the 26 (27%) IgG+/C3c- and 3 of the 33 (9%) IgG-/C3c- BP cases were positive for C4d. All 10 IgG+/C3c+ cases were also C4d positive. In the negative control group, 2 of the 39 (5%) were found positive for C4d. In conclusion, the current study shows that C4d is a more sensitive but not a 100% specific marker of BP. We conclude that C4d by DIF could be an interesting diagnostic adjunct for BP, particularly in IgG-/C3c- double negative cases.

Resting-State Functional Connectivity of the Punishment Network Associated With Conformity.

Fear of punishment prompts individuals to conform. However, why some people are more inclined than others to conform despite being unaware of any obvious punishment remains unclear, which means the dispositional determinants of individual differences in conformity propensity are poorly understood. Here, we explored whether such individual differences might be explained by individuals’ stable neural markers to potential punishment. To do this, we first defined the punishment network (PN) by combining all potential brain regions involved in punishment processing. We subsequently used a voxel-based global brain connectivity (GBC) method based on resting-state functional connectivity (FC) to characterize the hubs in the PN, which reflected an ongoing readiness state (i.e., sensitivity) for potential punishment. Then, we used the within-network connectivity (WNC) of each voxel in the PN of 264 participants to explain their tendency to conform by using a conformity scale. We found that a stronger WNC in the right thalamus, left insula, postcentral gyrus, and dACC was associated with a stronger tendency to conform. Furthermore, the FC among the four hubs seemed to form a three-phase ascending pathway, contributing to conformity propensity at every phase. Thus, our results suggest that task-independent spontaneous connectivity in the PN could predispose individuals to conform.

Association Analysis and Validation of Simple Sequence Repeat Markers for Fiber Fineness in Ramie (Boehmeria Nivea L. Gaudich)

ABSTRACT This study investigated simple sequence repeat (SSR) marker associated with fiber fineness in ramie (Boehmeria nivea L. Gaudich). Firstly, association analysis was carried out using 107 ramie accessions and 95 SSR markers. Three stable molecular markers related to fiber fineness were detected in multiple environments. Furthermore, we validated the three SSR markers using single marker association on a set of 286 accessions planted in another three environments. The result of polymorphisms analysis found that the average polymorphism information content (PIC) of three markers was 0.5149; average number of alleles was 3.6667; average genetic diversity was 0.6043; average Shannon-Wiener diversity index was 0.9710; and average major allele frequency (MAF) was 0.4488. We demonstrated that marker RAM39 was stably associated with fiber fineness, highlighting its utility for breeding. These findings provide a basis for genetic improvement of ramie quality traits through marker-assisted selection.

О волынском происхождении рукописного перевода корана на польский язык по рукописям литовских татар XVII-XХ веков

The author presents reasons in support of Ukrainian (Volhynian) origin of the translation of the Quran into Polish, written in Arabic script in the Tafsir created by Lithuanian Tatars and known in extant manuscript copies of the 17th-20th centuries, written in the Belarusian lands of the former Grand Duchy of Lithuania. The translation was made into the local regional variant of the Polish written language (polszczyzna kresowa), which is full of East Slavic linguistic features. This makes it possible to use the textually most stable (lexical and derivational) linguistic markers of the translation to determine the place of its origin: the adverb č i m a l o (‘quite a lot’) can be considered a Ukrainian-Belarusian common feature (originated in Ukraine), while the adjective r a n’ u s e n’ k y i (‘very early’) and the noun v i r š o v n y k (‘poet’) are actually Ukrainian. This indicates the emergence of the handwritten Polish translation of the Quran within the Tatar communities of Ukraine, from where it spread further north to Belarusian lands. The historical context allows us to link this Muslim translation with Ostroh as the oldest and largest Tatar centre of Volhynia and approximately date it to the turn of the 16th/17th centuries: between the publication of the Old Church Slavonic Ostroh Bible (1581) and the end of the active phase of the local Ostroh Academy (1608).

Species substitutions revealed through genotyping: Implications of traceability limitations and unregulated fishing

Trade flow of fisheries sector is very complex with several intermediaries and it is difficult to trace back the origin of a seafood commodity. Thus, traceability limitations could cause safety and quality issues in the final product delivered to the consumers. Decreased monitoring may also increase the possibilities of illegal, unreported and unregulated fishing practises. In global fisheries sector, a proper species identification system for identifying and preventing commercial frauds like species misrepresentation and illegal trade is mandatory. DNA analysis is a promising technique for food authentication as it provides increased specificity, sensitivity and reliable performance for accurate specimen identification and species confirmation. Mitochondrial cytochrome c oxidase subunit I gene (mtCOI) fragments represent one of the robust genetic marker for identification of specimens up to species level. It is a stable genetic marker which could be amplified from fresh, degraded, processed or cooked materials. India is having very limited regulations for preventing improper labelling of seafood items and ensuring authentication for traded fish and fishery products. This study focuses on the applicability of DNA barcodes over fish and fishery products traded at different sectors like local markets, supermarkets, restaurants etc. Samples collected from different stations of Ernakulam district (Kerala, S. India), were subjected to molecular analysis and COI sequences were developed. Among the 62 samples, 34 samples were identified as species substituents and the substitution rate was accounted up to 54.84%. In addition, trade of certain exotic/invasive and illegally cultured species were also confirmed. This study discusses the applicability of DNA barcoding in fisheries sector for preventing food fraud and suggests its implementation as a systematic regulatory programme conducted by governmental agencies for fishery stocks authentication.

Evaluation on the use of Nanopore sequencing for direct characterization of coronaviruses from respiratory specimens, and a study on emerging missense mutations in partial RdRP gene of SARS-CoV-2

Coronavirus disease 2019 (COVID-19) pandemic has been a catastrophic burden to global healthcare systems. The fast spread of the etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need to identify unknown coronaviruses rapidly for prompt clinical and public health decision making. Moreover, owing to the high mutation rate of RNA viruses, periodic surveillance on emerging variants of key virus components is essential for evaluating the efficacy of antiviral drugs, diagnostic assays and vaccines. These 2 knowledge gaps formed the basis of this study. In the first place, we evaluated the feasibility of characterizing coronaviruses directly from respiratory specimens. We amplified partial RdRP gene, a stable genetic marker of coronaviruses, from a collection of 57 clinical specimens positive for SARS-CoV-2 or other human coronaviruses, and sequenced the amplicons with Nanopore Flongle and MinION, the fastest and the most scalable massively-parallel sequencing platforms to-date. Partial RdRP sequences were successfully amplified and sequenced from 82.46% (47/57) of specimens, ranging from 75 to 100% by virus type, with consensus accuracy of 100% compared with Sanger sequences available (n = 40). In the second part, we further compared 19 SARS-CoV-2 RdRP sequences collected from the first to third waves of COVID-19 outbreak in Hong Kong with 22,173 genomes from GISAID EpiCoV™ database. No single nucleotide variants (SNVs) were found in our sequences, and 125 SNVs were observed from global data, with 56.8% being low-frequency (n = 1–47) missense mutations affecting the rear part of RNA polymerase. Among the 9 SNVs found on 4 conserved domains, the frequency of 15438G > T was highest (n = 34) and was predominantly found in Europe. Our data provided a glimpse into the sequence diversity of a primary antiviral drug and diagnostic target. Further studies are warranted to investigate the significance of these mutations.

Phylogenomic Analysis of Campylobacter fetus Reveals a Clonal Structure of Insertion Element ISCfe1 Positive Genomes.

Subspecies of the species Campylobacter fetus are associated with specific host niches including mammals and reptiles. Campylobacter fetus subsp. fetus is a zoonotic pathogen infecting humans. Infections can vary from an acute intestinal illness to severe systemic infections, with sheep and cattle as major reservoirs. In contrast, Campylobacter fetus subsp. venerealis causes bovine genital campylobacteriosis, which leads to abortion in cattle and a high economic burden for the farmers. Therefore, high-quality molecular subtyping is indispensable for interventional epidemiology. We used whole-genome sequencing (WGS) data of 283 Campylobacter fetus strains from 18 countries and compared several methods for Campylobacter fetus subtyping, including WGS, multilocus sequence typing, PCR assays, and the presence of the insertion element ISCfe1. We identified a highly clonal clade (designated as clade 1) that harbors the insertion sequence ISCfe1. The presence of this insertion sequence is an essential diagnostic tool for the identification of the subspecies Campylobacter fetus subsp. venerealis, serving as a target for several PCR assays. However, we have found a high sequence variability for the ISCfe1 besides the presence of ISCfe1-paralogues in certain other genomes (n = 7) which may cause incorrect diagnostic results. Clade 1 seems to be the cattle-specific clade of this species. We propose that only this clade might be designated as Campylobacter fetus subsp. venerealis as it harbors the ISCfe1 marker sequence, which is a major target for molecular methods currently used for Campylobacter fetus subspecies identification. Fostering this proposal, we defined eleven stable nucleotide markers specific for this clade. Additionally, we developed a bioinformatics toolbox for the fast identification of this clade based on WGS data. In conclusion, our results demonstrate that WGS can be used for Campylobacter fetus subtyping overcoming limitations of current PCR and MLST protocols.

Genetic diversity of major surface protein 1a of Anaplasma marginale in dairy cattle.

Major surface protein 1 alpha (Msp1α) is a stable genetic marker for identifying Anaplasma marginale. Our aims were to investigate the genetic diversity of A. marginale based on microsatellites and tandem repeats of the msp1α gene and to determine the phylogenetic relationships among six isolates obtained from 63 dairy cows positive for the pathogen from Turkey and from strains worldwide. Msp1α microsatellite analysis revealed infection with three genotypes (B, C, G). Genotype B was found in cows held in an intensive management system, while genotypes C and G were found in grazing cows. Microsatellite sequences produced SD-ATG distances of 19 and 23 nucleotides, with 23 nucleotides predominating, showing high infection potential. Fourteen different tandem repeats of A. marginale were found in the samples, and four msp1α gene repeats designated Tr4, Tr5, Tr6 and Tr7 were newly described. The majority of A. marginale isolates exhibited more than three Msp1a tandem repeats, with a maximum of seven. The Msp1a tandem repeats α-73-73-73-73-73-73 were commonly observed in intensively managed cows. The phylogenetic analysis revealed A. marginale strains from Turkey clustering with those of the Americas, Africa, Asia and Europe, suggesting a history of cattle trade and transport.

Correlation between malondialdehyde levels and disease activity in vitiligo

Background: Vitiligo is an acquired depigmented skin disease that occurs due to the death of melanocytes. There are various presumed theories about the cause of melanocyte death in vitiligo; one of them is oxidative stress theory. Lipid peroxidation is the primary manifestation of oxidative stress, producing malondialdehyde (MDA) as its end product. Malondialdehyde is a stable marker to assess the occurrence of oxidative stress. This study aims to prove the differences in serum MDA levels in the active vitiligo, stable vitiligo, and normal control groups.Method: This research is an analytical cross-sectional study involving 64 vitiligo patients with active and stable lesions and 20 normal controls. The study was conducted at Sanglah General Hospital Denpasar in February - November 2019. Serum MDA levels were measured to assess oxidative stress. The measurement of MDA levels was carried out by a spectrophotometer instrument using Competitive-ELISA method.Results: In this study, there were significant differences in MDA levels between active, stable vitiligo patients, and control group (p=0.000) with active vitiligo type have the highest MDA mean, followed by stable type and control group respectively.Conclusion: Increased serum MDA levels are an indicator of oxidative stress in vitiligo, which leads to melanocyte death and manifests as increased vitiligo disease activity. The results of this study support the role of oxidative stress in vitiligo pathogenesis and the use of oxidative stress indicators like serum MDA levels as biomarker in evaluating vitiligo disease activity.