Developing Next-Generation Live Attenuated Vaccines for Porcine Epidemic Diarrhea Using Reverse Genetic Techniques.

Porcine epidemic diarrhea virus (PEDV) is the etiology of porcine epidemic diarrhea (PED), a highly contagious digestive disease in pigs and especially in neonatal piglets, in which a mortality rate of up to 100% will be induced. Immunizing pregnant sows remains the most promising and effective strategy for protecting their neonatal offspring from PEDV. Although half a century has passed since its first report in Europe and several prophylactic vaccines (inactivated or live attenuated) have been developed, PED still poses a significant economic concern to the swine industry worldwide. Hence, there is an urgent need for novel vaccines in clinical practice, especially live attenuated vaccines (LAVs) that can induce a strong protective lactogenic immune response in pregnant sows. Reverse genetic techniques provide a robust tool for virological research from the function of viral proteins to the generation of rationally designed vaccines. In this review, after systematically summarizing the research progress on virulence-related viral proteins, we reviewed reverse genetics techniques for PEDV and their application in the development of PED LAVs. Then, we probed into the potential methods for generating safe, effective, and genetically stable PED LAV candidates, aiming to provide new ideas for the rational design of PED LAVs.

Techniques for assessing telomere length: A methodological review.

Telomeres are located at the ends of chromosomes and have specific sequences with a distinctive structure that safeguards genes. They possess capping structures that protect chromosome ends from fusion events and ensure chromosome stability. Telomeres shorten in length during each cycle of cell division. When this length reaches a certain threshold, it can lead to genomic instability, thus being implicated in various diseases, including cancer and neurodegenerative disorders. The possibility of telomeres serving as a biomarker for aging and age-related disease is being explored, and their significance is still under study. This is because post-mitotic cells, which are mature cells that do not undergo mitosis, do not experience telomere shortening due to age. Instead, other causes, for example, exposure to oxidative stress, can directly damage the telomeres, causing genomic instability. Nonetheless, a general agreement has been established that measuring telomere length offers valuable insights and forms a crucial foundation for analyzing gene expression and epigenetic data. Numerous approaches have been developed to accurately measure telomere lengths. In this review, we summarize various methods and their advantages and limitations for assessing telomere length.

MLPA in the initial genetic screening of patients with acute myeloid leukemia.

INTRODUCTION: This study aimed to assess the effectiveness of multiplex ligase-dependent probe amplification (MLPA) in the initial genetic screening of patients with acute myeloid leukemia (AML) since current risk stratification and clinical management depend on molecular-genetic markers. METHODS: We performed a prospective case-control study on newly diagnosed patients from the Clinical hematology clinic of UMHAT "St. Marina", Varna, for the period 02.2022 - 02.2023. MLPA - a semiquantitative PCR-based method, was implemented with probes for 40 AML/myelodysplastic syndrome-typical genetic changes. We compared these findings with a parallelly carried out cytogenetic analysis, part of the routine diagnostic process. RESULTS: We assessed 61 patients - 29 females and 32 males, median age of 61 years for females and 65 for males (min-max 20-89). 34 (56%) of all showed pathological results, while the rest 27 (44%) did not. Of the 34, 22 (65%) had a single gene variant in genes NPM1, DNMT3A, FLT3, and IDH2, isolated or in combination. 18 (53%) of the same 34 also had copy number aberration (CNA) in chromosomes 4, 5, 6, 7, 11, 14, 17, and 21. The latter were either isolated or in combination with other findings. 8 of the 18 cases also underwent cytogenetic analysis, with concordance between the two methods in 4. CONCLUSION: MLPA is an informative method for initial genetic assessment in addition to cytogenetic analysis. Still, more patients are needed to draw finite conclusions on its eligibility for routine use. Given the significant percentage of normal results - 44%, simultaneous evaluation of more genetic markers, included in current guidelines, is reasonable.

Review: Wildlife forensic genetics-Biological evidence, DNA markers, analytical approaches, and challenges.

Wildlife-related crimes are the second most prevalent lawbreaking offense globally. This illicit trade encompasses hunting, breeding and trafficking. Besides diminishing many species and their habitats and ecosystems, hindering the economic development of local communities that depend on them, undermining the rule of law and financing terrorism, various cross-species transmissions (zoonoses) of pathogens, including COVID-19, can be attributed to wildlife crimes. Wildlife forensics applies interdisciplinary scientific analyses to support law enforcement in investigating wildlife crimes. Its main objectives are to identify the taxonomic species in question, determine if a crime has been committed, link a suspect to the crime and support the conviction and prosecution of the perpetrator. This article reviews wildlife crime and its implications, wildlife forensic science investigation, common forms of wildlife biological evidence, including DNA, wildlife DNA techniques and challenges in wildlife forensic genetics. The article also reviews the contributions of genetic markers such as short tandem repeat (STR) and mitochondrial DNA (mtDNA) markers, which provide the probative genetic data representing the bulk of DNA evidence for solving wildlife crime. This review provides an overview of wildlife DNA databases, which are critical for searching and matching forensic DNA profiles and sequences and establishing how frequent forensic DNA profiles and sequences are in a particular population or geographic region. As such, this review will contain an in-depth analysis of the current status of wildlife forensic genetics, and it will be of general interest to wildlife and conservation biologists, law enforcement officers, and academics interested in combating crimes against wildlife using animal forensic DNA methods.

Rainbow smelt (Osmerus mordax) influence on walleye (Sander vitreus) recruitment decline: mtDNA evidence supporting the predation hypothesis.

Rainbow smelt (Osmerus mordax) have been introduced widely but are associated with declines in walleye (Sander vitreus) recruitment. A primary hypothesis for these declines is that O. mordax consume larval S. vitreus. We confirmed overlapping spatial-temporal distributions of larval S. vitreus and O. mordax in our study system and used mtDNA analyses to determine if O. mordax stomach contents contained S. vitreus. Approximately 20% of O. mordax composite stomach samples were considered positive for S. vitreus consumption. These findings support the predation hypothesis and have S. vitreus management/stocking implications.

Study on the Prevalence of Pneumocystis jirovecii as a Causative Agent of Lung Pathology in People with Different Immune Status.

BACKGROUND: Pneumocystis pneumonia (PCP) commonly affects immunocompromised individuals, whereas in immunocompetent persons, it occurs relatively rarely, and in most cases, the Pneumocystis infection is detected as an asymptomatic colonization. The present study aimed to establish the prevalence of Pneumocystis jirovecii infection in human hosts with different immune status (immunocompromised and immunocompetent), using molecular diagnostic methods, and to compare their diagnostic value with that of classical staining methods. METHODS: We used the collected-to-this-moment data from a prospective study on the prevalence of pneumocystosis among the Bulgarian population. Clinical specimens (including throat secretions, induced sputum, tracheal aspirates, and bronchoalveolar lavage) collected from 220 patients suspected of PCP (153 immunocompetent and 67 immunocompromised patients) were examined with microscopic staining methods and real-time PCR for detection of P. jirovecii. Results: DNA of the pathogen was detected in 38 (17%) specimens (32 immunocompromised patients and 6 immunocompetent subjects). From all 220 clinical samples examined by staining methods, only five (2%) P. jirovecii cysts were detected by the Gomori stain. All patients with PCP were treated with trimethoprim-sulfamethoxazole, but in ten of them (HIV-positive patients), the disease had a fatal outcome. CONCLUSIONS: This study is the first in Bulgaria including the main available laboratory methods for diagnosis of human pneumocystosis. Regarding the etiological diagnosis of PCP, in our study the sensitivity of real-time PCR was higher compared to the staining methods. The choice of a method for sample collection and examination has an important role in the efficiency of the laboratory diagnostics.

A robotic system for automated genetic manipulation and analysis of Caenorhabditis elegans.

The nematode Caenorhabditis elegans is one of the most widely studied organisms in biology due to its small size, rapid life cycle, and manipulable genetics. Research with C. elegans depends on labor-intensive and time-consuming manual procedures, imposing a major bottleneck for many studies, especially for those involving large numbers of animals. Here, we describe a general-purpose tool, WormPicker, a robotic system capable of performing complex genetic manipulations and other tasks by imaging, phenotyping, and transferring C. elegans on standard agar media. Our system uses a motorized stage to move an imaging system and a robotic arm over an array of agar plates. Machine vision tools identify animals and assay developmental stage, morphology, sex, expression of fluorescent reporters, and other phenotypes. Based on the results of these assays, the robotic arm selectively transfers individual animals using an electrically self-sterilized wire loop, with the aid of machine vision and electrical capacitance sensing. Automated C. elegans manipulation shows reliability and throughput comparable with standard manual methods. We developed software to enable the system to autonomously carry out complex protocols. To validate the effectiveness and versatility of our methods, we used the system to perform a collection of common C. elegans procedures, including genetic crossing, genetic mapping, and genomic integration of a transgene. Our robotic system will accelerate C. elegans research and open possibilities for performing genetic and pharmacological screens that would be impractical using manual methods.

Additional value of red blood cell parameters in predicting uncommon α-thalassemia; experience from 10 years of α-globin gene sequencing and copy number variation analysis.

INTRODUCTION: The diagnosis of rare forms of α-thalassemia requires laborious genetic analyses. Accurate sample selection for such evaluation is therefore essential. The main objectives of this study were to investigate the predictive power of red blood cell parameters to detect rare forms of α-thalassemia (substudy 1), and to explore the frequency of rare versus common forms of α-thalassemia in our sample population (substudy 2). METHODS: In substudy 1, we reviewed all blood samples selected for extended α-hemoglobinopathy evaluation at our laboratory during 2011-2020 (n = 1217), which included DNA sequencing and/or copy number variation analysis. We assessed α-thalassemia positive samples at different levels of mean corpuscular hemoglobin (MCH) alone and in combination with results for red blood cell count (RBC) or red cell distribution width (RDW). In substudy 2, we examined the distribution of α-thalassemia genotypes for all samples submitted to a first-tier hemoglobinopathy evaluation at our laboratory during 2014-2020 (n = 6495). RESULTS: In substudy 1, both RBC and RDW added predictive value in detecting rare forms of α-thalassemia in samples from adults and children. In adult samples with MCH ≤ 23 pg, the presence of erythrocytosis increased the detection rate from 27% to 74% as compared to non-erythrocytosis, while normal RDW increased the detection rate from 36% to 86% as compared to elevated RDW. In substudy 2, rare forms of α-thalassemia were detected in 12% of α-thalassemia positive samples. CONCLUSION: Initial assessment of MCH, RBC, and RDW provided valuable predictive information about the presence of rare forms of α-thalassemia during hemoglobinopathy evaluation.

Arousal mechanism after sevoflurane anesthesia in rats: orexinergic modulation in dorsal raphe nucleus by optogenetic and chemogenetic techniques / 中华麻醉学杂志

Objective:To investigate the arousal mechanism after sevoflurane anesthesia using orexinergic modulation in dorsal raphe nucleus(DRN) by optogenetic and chemogenetic techniques in rats.Methods:Forty-five healthy male Hcrt-Cre rats, aged 10-12 weeks, weighing 220-250 g, were divided into 6 groups by the random number table method: optical-excitatory group (CHR2 group, n=5), optical-inhibitory group (eNpHR group, n=5), optical-control group (O-CON group, n=5); chemogenetic-excitatory group (hm3Dq group, n=10), chemogenetic-inhibitory group (hm4Di group, n=10) and chemogenetic-control group (C-CON group, n=10). The optogenetic or chemogenetic techniques were used in each group. Three weeks after injecting the rat virus, anesthesia was induced and maintained with 2.7% sevoflurane anesthesia in 1.5 L/min O 2, and the EEG data were continuously recorded throughout the process. The burst suppression ratio (%BSR) was recorded at 2 min before and of laser stimulation. Combining optogenetic and chemogenetic strategies, it was investigated that whether activation of orexinergic projection to DRN could modulate anesthetic behaviors during sevoflurane anesthesia. Results:Compared with C-CON group, the recovery of righting reflex (RORR) time was significantly shortened after sevoflurane anesthesia in hm3Dq group ( P<0.05), and the RORR time was significantly prolonged after sevoflurane anesthesia in hm4Di group and eNpHR group ( P<0.05). Compared with O-CON group or the baseline at 2 min before light stimulation, the %BSR was significantly decreased during 473nm laser stimulation in CHR2 group ( P<0.05), and no statistically significant change was found in the %BSR during 473nm laser stimulation in eNpHR group ( P>0.05). Compared with O-CON group, the RORR time was significantly shortened after sevoflurane anesthesia in CHR2 group ( P<0.05). Conclusions:Lateral hypothalamic area orexin-DRN neural circuit plays a key role in promoting arousal from general anesthesia in rats.

Chip-based digital Polymerase Chain Reaction as quantitative technique for the detection of PIK3CA mutations in breast cancer patients.

Background: PIK3CA is a gene frequently mutated in breast cancer. With the FDA approval of alpelisib, the evaluation of PIK3CA for activating mutations is becoming routinely. Novel platforms for gene analysis as digital PCR (dPCR) are emerging as a potential replacement for the traditional Sanger sequencing. However, there are still few studies on chip-based dPCR to detect mutations in tumor samples. Thus, this cross-sectional study aimed to assess the sensibility of a chip-based dPCR to detect and quantify PIK3CA mutations and compare its performance with Sanger sequencing. Materials and Methods: Tumor samples from 57 breast cancer patients (22 pre-treatment samples, 32 tumors after neoadjuvant chemotherapy, and three lymph nodes) were collected and analyzed by Sanger sequencing and dPCR for the three PIK3CA most relevant mutations (p.E545K, p. H1047R, and p. H1047L). Digital PCR sensitivity, specificity, and overall performance were estimated by contingency tables, receptor operator characteristic (ROC), and area under the curve (AUC). Association of PIK3CA mutations with clinicopathological variables was conducted. Results: Sanger sequencing identified PIK3CA mutations in six patients (10.5%), two with p. H1047R, and four with p. E545K. Digital PCR confirmed those mutations and identified 19 additional patients with at least one mutation. Comparison between dPCR and Sanger sequencing showed a sensitivity of 100% (95% CI 53-100%), and a specificity of 84.2% (95% CI 83-84.2%). Besides, p. H1047R mutation detected by dPCR showed a significant association with breast cancer phenotype (p = 0.019) and lymphatic nodes infiltration (p = 0.046). Conclusions: Digital PCR showed a high sensitivity to detect mutations in tumor samples and it might be capable to detect low-rate mutations and tumor subpopulations not detected by Sanger sequencing.

Bats and pollution: Genetic approaches in ecotoxicology.

Environmental pollution drives the decline of species and, as flying mammals, bats can be considered to be excellent indicators of environmental quality, and the analysis of genetic biomarkers in these animals can provide important parameters for the assessment of environmental health. This review verifies the trends in pollution research, in particular, the use of genetic markers in the study of bats, based on a literature search of the Web of Science and Scopus platforms. Sixteen publications were identified during the search, which focused on the timeframe between 1996 and March 2022, including investigations of the effects of heavy metals, agricultural pesticides, and radiation. The studies were based primarily on the application of biomarkers for genotoxic analysis, including the comet assay, micronucleus test, and the Polymerase Chain Reaction (PCR). Only 55 bat species have been investigated up to now, that is, 4% of the 1447 currently recognized. In general, bats exposed to polluted environments presented a higher frequency of genotoxic and mutagenic damage than those sampled in clean environments. Given the importance of the diverse ecological functions provided by bats, including pest control, pollination, and seed dispersal, it is increasingly necessary to investigate the damage caused to the health of these animals exposed to areas with high concentrations of contaminants. Although genetic biomarkers have been used to investigate physiological parameters in bats for more than two decades, then, many knowledge gaps remain, worldwide, in terms of the number of species and localities investigated.

Introducing stimulogenetics, unraveling pertinent semantic ambiguity, and determining clinical relevance among novel neuromodulation strategies.

Deep brain stimulation involving the stereotactic implantation of electrodes in the deeper neural tissue remains one of the most trusted nonpharmacotherapeutic approaches for neuromodulation in the clinical setting. The recent advent of techniques that can modulate the neural structure and/or function at the cellular level has stimulated the exploration of these strategies in managing neurological and psychiatric disorders. Optogenetics, which is widely employed in experimental research, is the prototype of the above techniques. Other methods such as chemogenetics, sonogenetics, and magnetogenetics have also been introduced. Although these strategies possess several noticeable differences, they have an overlapping conceptual framework enabling their classification under a singular hypernym. This article introduces this hypernym, "stimulogenetics" in an attempt to solve the pertinent ambiguity to aid the classification of existing literature. The article also compares the strategies classified under stimulogenetics and concludes that the current literature suggests that nonsurgical approaches such as chemogenetics and sonogenetics are better suited for clinical applications. However, due to the dearth of clinical studies, it is not possible to determine this definitively.

Clinical validation of engineered CRISPR/Cas12a for rapid SARS-CoV-2 detection.

Background: The coronavirus disease (COVID-19) caused by SARS-CoV-2 has swept through the globe at an unprecedented rate. CRISPR-based detection technologies have emerged as a rapid and affordable platform that can shape the future of diagnostics. Methods: We developed ENHANCEv2 that is composed of a chimeric guide RNA, a modified LbCas12a enzyme, and a dual reporter construct to improve the previously reported ENHANCE system. We validated both ENHANCE and ENHANCEv2 using 62 nasopharyngeal swabs and compared the results to RT-qPCR. We created a lyophilized version of ENHANCEv2 and characterized its detection capability and stability. Results: Here we demonstrate that when coupled with an RT-LAMP step, ENHANCE detects COVID-19 samples down to a few copies with 95% accuracy while maintaining a high specificity towards various isolates of SARS-CoV-2 against 31 highly similar and common respiratory pathogens. ENHANCE works robustly in a wide range of magnesium concentrations (3 mM-13 mM), allowing for further assay optimization. Our clinical validation results for both ENHANCE and ENHANCEv2 show 60/62 (96.7%) sample agreement with RT-qPCR results while only using 5 µL of sample and 20 minutes of CRISPR reaction. We show that the lateral flow assay using paper-based strips displays 100% agreement with the fluorescence-based reporter assay during clinical validation. Finally, we demonstrate that a lyophilized version of ENHANCEv2 shows high sensitivity and specificity for SARS-CoV-2 detection while reducing the CRISPR reaction time to as low as 3 minutes while maintaining its detection capability for several weeks upon storage at room temperature. Conclusions: CRISPR-based diagnostic platforms offer many advantages as compared to conventional qPCR-based detection methods. Our work here provides clinical validation of ENHANCE and its improved form ENHANCEv2 for the detection of COVID-19.

Blockade of TGF-ß (Transforming Growth Factor Beta) Signaling by Deletion of Tgfbr2 in Smooth Muscle Cells of 11-Month-Old Mice Alters Aortic Structure and Causes Vasomotor Dysfunction-Brief Report.

BACKGROUND: To test the hypothesis that smooth muscle cell (SMC) TGF-ß (transforming growth factor beta) signaling contributes to maintenance of aortic structure and function beyond the early postnatal period. METHODS: We deleted the TBR2 (type 2 TGF-ß receptor) in SMC of 11-month-old mice (genotype Acta2-CreERT2+/0Tgfbr2f/f, termed TBR2SMΔ) and compared their ascending aorta structure and vasomotor function to controls (Acta2-CreERT20/0Tgfbr2f/f, termed TBR2f/f). RESULTS: We confirmed loss of aortic SMC TBR2 by immunoblotting. Four weeks after SMC TBR2 loss, TBR2SMΔ mice did not have aortic rupture, ulceration, dissection, dilation, or evidence of medial hemorrhage. However, aortic medial area of TBR2SMΔ mice was increased by 27% (0.14±0.01 versus 0.11±0.01 mm2; P=0.01) and medial thickness was increased by 23% (40±1.9 versus 33±1.3 µm; P=0.004) compared with littermate controls. Wire myography performed on ascending aortic rings showed hypercontractility of TBR2SMΔ aortas to phenylephrine (Emax, 15.9±1.2 versus 10.8±0.7 mN; P=0.0003) and reduced relaxation and sensitivity to acetylcholine (Emax, 64±14% versus 96±2%; P=0.001; -logEC50, 6.9±0.1 versus 7.7±0.1; P=0.0001). Neither maximal relaxation nor sensitivity to sodium nitroprusside differed (Emax, 102±0.3% versus 101±0.3%; -logEC50, 8.0±0.04 versus 7.9±0.08; P>0.4 for both). CONCLUSIONS: Loss of TGF-ß signaling in aortic SMC of 1-year-old mice does not cause early severe aortopathy or death; however, it causes mild structural and substantial physiological abnormalities. SMC TGF-ß signaling plays an important role in maintaining aortic homeostasis in older mice. This role should be considered in the design of clinical studies that aim to prevent aortopathy by blocking SMC TGF-ß signaling.

Diagnostic performance of RT-PCR-based sample pooling strategy for the detection of SARS-CoV-2.

BACKGROUND: The rapid spread of SARS-CoV-2 has created a shortage of supplies of reagents for its detection throughout the world, especially in Latin America. The pooling of samples consists of combining individual patient samples in a block and analyzing the group as a particular sample. This strategy has been shown to reduce the burden of laboratory material and logistical resources by up to 80%. Therefore, we aimed to evaluate the diagnostic performance of the pool of samples analyzed by RT-PCR to detect SARS-CoV-2. METHODS: A cross-sectional study of diagnostic tests was carried out. We individually evaluated 420 samples, and 42 clusters were formed, each one with ten samples. These clusters could contain 0, 1 or 2 positive samples to simulate a positivity of 0, 10 and 20%, respectively. RT-PCR analyzed the groups for the detection of SARS-CoV-2. The area under the ROC curve (AUC), the Youden index, the global and subgroup sensitivity and specificity were calculated according to their Ct values that were classified as high (H: ≤ 25), moderate (M: 26-30) and low (L: 31-35) concentration of viral RNA. RESULTS: From a total of 42 pools, 41 (97.6%) obtained the same result as the samples they contained (positive or negative). The AUC for pooling, Youden index, sensitivity, and specificity were 0.98 (95% CI, 0.95-1); 0.97 (95% CI, 0.90-1.03); 96.67% (95% CI; 88.58-100%) and 100% (95% CI; 95.83-100%) respectively. In the stratified analysis of the pools containing samples with Ct ≤ 25, the sensitivity was 100% (95% CI; 90-100%), while with the pools containing samples with Ct ≥ 31, the sensitivity was 80% (95% CI, 34.94-100%). Finally, a higher median was observed in the Ct of the clusters, with respect to the individual samples (p < 0.001). CONCLUSIONS: The strategy of pooling nasopharyngeal swab samples for analysis by SARS-CoV-2 RT-PCR showed high diagnostic performance.

Acute myelogenous leukemia - current recommendations and approaches in molecular-genetic assessment.

Acute myelogenous leukemia is a multi-step hematological malignancy, affecting function, growth, proliferation and cell cycle of myeloid precursors. Overall assessment of patients with the disease requires among everything else, a comprehensive investigation of the genetic basis through various methods such as cytogenetic and molecular-genetic ones. This clarification provides diagnostic refinement and carries prognostic and predictive value in respect of essential therapeutic choices.With this review of the literature, we focus on summarizing the latest recommendations and preferred genetic methods, as well as on emphasizing on their general benefits and limitations. Since none of these methods is actually totipotent, we also aim to shed light over the often-difficult choice of appropriate genetic analyses.

Lessons learnt from multifaceted diagnostic approaches to the first 150 families in Victoria's Undiagnosed Diseases Program.

BACKGROUND: Clinical exome sequencing typically achieves diagnostic yields of 30%-57.5% in individuals with monogenic rare diseases. Undiagnosed diseases programmes implement strategies to improve diagnostic outcomes for these individuals. AIM: We share the lessons learnt from the first 3 years of the Undiagnosed Diseases Program-Victoria, an Australian programme embedded within a clinical genetics service in the state of Victoria with a focus on paediatric rare diseases. METHODS: We enrolled families who remained without a diagnosis after clinical genomic (panel, exome or genome) sequencing between 2016 and 2018. We used family-based exome sequencing (family ES), family-based genome sequencing (family GS), RNA sequencing (RNA-seq) and high-resolution chromosomal microarray (CMA) with research-based analysis. RESULTS: In 150 families, we achieved a diagnosis or strong candidate in 64 (42.7%) (37 in known genes with a consistent phenotype, 3 in known genes with a novel phenotype and 24 in novel disease genes). Fifty-four diagnoses or strong candidates were made by family ES, six by family GS with RNA-seq, two by high-resolution CMA and two by data reanalysis. CONCLUSION: We share our lessons learnt from the programme. Flexible implementation of multiple strategies allowed for scalability and response to the availability of new technologies. Broad implementation of family ES with research-based analysis showed promising yields post a negative clinical singleton ES. RNA-seq offered multiple benefits in family ES-negative populations. International data sharing strategies were critical in facilitating collaborations to establish novel disease-gene associations. Finally, the integrated approach of a multiskilled, multidisciplinary team was fundamental to having diverse perspectives and strategic decision-making.

Intervenciones genéticas de Aedes aegypti para el control de arbovirosis / Genetic interventions of Aedes aegypti for the control of arboviruses

La ineficacia de las estrategias actuales para el control químico de los mosquitos vectores plantea la necesidad de desarrollar enfoques novedosos, entre estos están las estrategias genéticas para reducir las poblaciones de mosquitos vectores o sustituirlos por aquellos que no son capaces de transmitir patógenos, esto se logra a través de herramientas moleculares que permiten la manipulación y transgénesis de genes. Las secuencias del genoma de los mosquitos y las bases de datos de marcadores de secuencias expresadas asociadas permiten investigaciones a gran escala para proporcionar nuevos conocimientos sobre las vías evolutivas, bioquímicas, genéticas, metabólicas y fisiológicas. Además, la genómica comparativa revela las bases de los mecanismos evolutivos con especial atención a las interacciones específicas entre vectores y patógenos. Se ha desarrollado tecnología de transgénesis para el mosquito de la fiebre amarilla y dengue, Aedes aegypti. Se ha logrado integración exitosa de ADN exógeno en la línea germinal de este mosquito con los elementos transponibles. La disponibilidad de múltiples elementos y genes marcadores proporciona un poderoso conjunto de herramientas para investigar las propiedades biológicas básicas de este insecto vector, así como los materiales para desarrollar nuevas estrategias de control genético de poblaciones de mosquitos basadas en la técnica del insecto estéril. Una de estas estrategias consiste en liberar a la población machos esterilizados por radiación; otro, de integrar un gen letal dominante bajo el control de un promotor específico en hembras inmaduras. El uso de esta técnica de modificación genética constituirá una herramienta importante para el manejo integrado de vectores(AU)

The ineffectiveness of current strategies for the chemical control of vector mosquitoes raises the need to develop novel approaches, among these are genetic strategies to reduce populations of vector mosquitoes or replace them with those that are not capable of transmitting pathogens, this is achieved through molecular tools that allow the manipulation and transgenesis of genes. Mosquito genome sequences and associated expressed sequence marker databases enable large-scale investigations to provide new insights into evolutionary, biochemical, genetic, metabolic, and physiological pathways. Furthermore, comparative genomics reveals the basis of evolutionary mechanisms with special attention to the specific interactions between vectors and pathogens. Transgenesis technology has been developed for the yellow fever and dengue mosquito, Aedes aegypti. Successful integration of exogenous DNA into the germ line of this mosquito with the transposable elements has been achieved. The availability of multiple elements and marker genes provides a powerful set of tools to investigate the basic biological properties of this insect vector, as well as the materials to develop new strategies for genetic control of mosquito populations based on the sterile insect technique. One of this strategy is to release radiation-sterilized males into the population; another, to integrate a dominant lethal gene under the control of a specific promoter in immature females. The use of this genetic modification technique will constitute an important tool for the integrated management of vectors(AU)

Elevated IgA and IL-10 levels in very-early-onset inflammatory bowel disease secondary to IL-10 receptor deficiency / Níveis elevados de IgA e IL-10 na doença inflamatória intestinal de início muito precoce secundária à deficiência do receptor de IL-10

ABSTRACT Objective: To report two patients with very-early-onset inflammatory bowel disease (VEOIBD) secondary to interleukin-10 receptor (IL-10R) mutations, explore immunophenotyping data and plasma cytokine profile on these cases compared to healthy controls, and describe the phenotype of IL-10/IL-10R mutations based on a literature review. Case description: We report on two female infants referred to our tertiary center at the age of ten months, with severe colonic and perianal disease, as well as significant malnutrition, who had shown limited response to usual inflammatory bowel disease (IBD) therapy agents. In the first case, whole-exome sequencing (WES) revealed a homozygous (c.537G>A/p.T179T) mutation in exon 4 of the IL-10RA gene, while in the second patient, compound heterozygosity was identified, also in the IL-10RA gene (chr11:117.859.199 variant A>G/p.Tyr57Cys and chr11: 117.860.335 variant G>T/p.Val123Leu). Both patients underwent hematopoietic cell transplantation (HCT). Immunological work-up of these patients revealed increased IL-10 plasma levels and increased IgA. Comments: Our case reports disclose novel findings on plasma cytokine profile in IL-10R deficiency, and we describe the severe phenotype of IL-10/IL-10R deficiency that should be recognized by physicians.

RESUMO Objetivo: Relatar os casos de duas pacientes com doença inflamatória intestinal de início muito precoce (em inglês VEOIBD) secundária a mutações do receptor de interleucina 10 (IL-10R), explorar dados de imunofenotipagem e perfil de citocinas plasmáticas nesses casos em comparação com indivíduos saudáveis e descrever o fenótipo de mutações IL-10/IL-10R com base em uma revisão da literatura. Descrição do caso: Duas lactentes do sexo feminino foram encaminhadas ao nosso centro terciário, ambas com dez meses no momento do encaminhamento, com doença colônica e perianal grave, bem como desnutrição significativa, tendo uma resposta limitada aos agentes de terapia usuais de doença inflamatória intestinal (DII). No primeiro caso, o sequenciamento completo do exoma revelou mutação homozigótica (c. 537G>A/p.T179T) no exon 4 do gene IL-10RA, enquanto no segundo caso heterozigosidade composta foi identificada também no gene IL-10RA [chr11: 117.859.199 - variante A>G/p.Tyr57Cys e chr11: 117.860.335 - variante G>T/ p.Val123Leu]. Ambas as pacientes foram submetidas a Transplante de Células-Tronco Hematopoiéticas. A investigação imunológica das pacientes revelou aumento dos níveis plasmáticos de IL-10 e aumento da IgA. Comentários: Nossos relatos de casos descrevem novos achados no perfil de citocinas plasmáticas na deficiência de IL-10R, e relatamos o fenótipo grave da deficiência de IL-10/IL-10R que deve ser reconhecido pelos médicos.