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Background: Differences in the embryonic development of the colonic mucosa determine the physiological embryonic-anatomical asymmetry of its structure and can manifest themselves via different molecular phenotypes (expression profiles) of the colon segments. These molecular characteristics are hypothesized to determine differences in the carcinogenesis mechanisms and influence the prognosis of right- or left-sided colorectal cancer (CRC). Studies of the tumors molecular phenotypes depending on their localization may be of interest for assessment of the prognosis and choice of treatment for CRC. Aim: To perform comparative analysis of molecular phenotypes of the normal colonic mucosa and adenocarcinoma CRC tissues depending on the natural embryonic anatomic asymmetry of the colon. Materials and methods: We performed a retrospective study of molecular phenotypes (mRNA expression of 61 genes) from different embryonic-anatomical parts of healthy colon and CRC. The normal group included 254 samples of mucosa from three different parts of the colon from 74 healthy donors who had no cancer and no organic abnormalities of the colon, including 90 samples from the right colon, 116 from the left colon, and 48 from the rectum. The CRC group consisted of 154 samples of localized stage T1–4N0–2M0 adenocarcinoma from 154 patients who had not received neoadjuvant radio- and chemotherapy, including 40 samples from the right colon, 54 from the left colon, and 60 from the rectum. The relative mRNA abundance of 61 genes was assessed by reverse-transcriptase polymerase chain reaction. In both groups, the resulting expression phenotypes were compared between the anatomical parts of the colon. Statistical management of the data included the discriminant analysis with stepwise inclusion of variables. Results: Based on the assessment of the mRNA level of the studied genes, a discriminant model was built that allows for classification of the normal group samples according to their anatomic origin in the colon with an accuracy of 95.8%. The most significant (p 0.05) for classification are the following 19 genes: CCND1, SCUBE2, TERT, BAG1, NDRG, IL1b, IL2Ra, IL7, ESR1, TGFb, IGF1, MMP9, MMP11, PAPPA, CD45, CD69, TLR2, TLR4, LIFR. The discriminant model built for the CRC group included 27 genes and made it possible to differentiate samples from three parts of colon with an accuracy of 75.2%. A statistically significant (p 0.05) contribution to the samples differentiation by the discriminant model was made by the COX-2, BIRC5, LIFR, TPA, IL1b, MMP11, MMP7, and P16INK4A genes. When combining samples from the two groups into one model in accordance with their embryonic-anatomical origin, there was a clear separation of tumor tissue samples and healthy colonic mucosa in the discriminant function space. Conclusion: The analysis of CRC gene expression profiles using the discriminant model showed that genetic changes in the colonic mucosa in CRC flatten the molecular phenotypic boundaries of the embryonic-anatomical parts. These changes are specific to CRC, forming a particular “pathological” molecular phenotype.

BACKGROUND: Hepatitis C virus (HCV) infection, predominantly transmitted by exposure to infected blood, remains one of the major public health problems worldwide. This study aims to identify the risk factors of HCV transmission and its chronic complications among the study group. MATERIALS AND METHODS: This retrospective study was approved by the Research and Ethical Review and Approve Committee (RERAC) of Oman and conducted at a secondary-care hospital situated in the North Batinah region of Oman. The study population included all HCV cases confirmed by positive serology and reverse-transcription polymerase chain reaction tests during their presence at the hospital between January 2017 and December 2022. The relevant data of the study population were retrieved from the hospital electronic health record system. The data were analyzed using the Statistical Package for the Social Sciences (SPSS), Version 26.0. RESULTS: A total of 177 HCV confirmed cases were included in the study. HCV infection was predominant among males (74%) and individuals of the age group of 21–60 years (74.6%). Genotyping was possible only in 107 cases. Among HCV genotypes, genotype 3 (58.9%) was the most frequently identified, followed by genotype 1 (34.6%). Hemodialysis (21.5%), history of blood transfusion (16.4%), and injection drug use (11.9%) were the major risk factors for HCV infection, while cirrhosis (7.3%) and fatty liver disease (4%) were the most frequently observed chronic HCV complications. HCV infection in the spouse/partner (21.5%), alcohol use (7.3%), and co-infection with hepatitis B virus (2.3%) and human immunodeficiency virus (1.7%) were the other significant factors detected in our study population. CONCLUSIONS: HCV is a multi-factorial disease leading to severe chronic complications, thus representing a public health threat. This clearly emphasizes the cruciality of HCV community awareness campaigns and enhancement of Omani national guidelines for early screening of high-risk groups as well as effective management of HCV-infected cases to reduce the substantial burden of the disease on patients as well as the healthcare system.

Background: COVID-19 pandemic has stirred an unexpected turmoil in health care worldwide. The strategic conduct of critical care would warrant an effective preparedness plan, thorough knowledge of the disease manifestations, and relevant therapeutic strategy to sail through this crisis. Methods: English medical literature with MeSh database was searched using the key words such as “COVID-19 pandemic,” “ICU preparedness,” “COVID-19 management,” “COVID-19 therapy,” and “COVID-19 systemic effects.” The relevant studies were included with significant inputs from interdepartmental meetings for the formulation of a plan. Results: The analysis of the respective studies highlighted the requirements of COVID-19 designated intensive care units (ICUs) with special provisions and the therapeutic agents being used in critical patients with emphasis on Remdesivir, Dexamethasone, Convalescent Plasma, and insight into newer agents. The systemic manifestations of COVID-19 requiring ICU care such as acute respiratory distress syndrome, myocardial injury, arrhythmias, hypercoagulable state, and acute renal dysfunction have been highlighted. The need of updating records with research protocols cannot be disregarded. The care of patients should not compromise the health-care personnel requirements. Conclusion: The evidence-based preparedness strategy can curtail the critical care crunch in COVID-19 management; however, institutional specific approach should be formulated.

Coronavirus disease 2019 (COVID‐19) has been shown to be associated with a lot of neurological complications, of whom Guillain‐Barre syndrome (GBS) is an important post‐infectious consequentiality. More than 220 patients with GBS have been reported thus far. We intend to share our experience with five patients of GBS where one of them had severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in the cerebrospinal fluid (CSF). This is the first‐ever report demonstrating the presence of SARS‐CoV‐2 in the CSF of an adult patient; a similar occurrence has recently been described in a pediatric patient. We wish to emphasize the fact that commonly GBS occurs as a result of a post‐infectious process but in a few cases where the symptoms of COVID‐19 and GBS occur concurrently, corresponding to the viremic phase, separate pathogenesis needs to be thought of. This para‐infectious nature is exemplified by the presence of virus in the cerebrospinal fluid of one of our patients. We review the neuroinvasive potential of SARS‐Cov‐2 in this regard and draw parallels with Cytomegalovirus, Zika virus, and Human Immunodeficiency virus‐associated occurrences of GBS. [ABSTRACT FROM AUTHOR]

To investigate the presence of SARS-CoV-2 RNA in tears of patients with moderate to severe coronavirus disease 2019 (COVID-19). Cross-sectional study. Patients with laboratory-proven moderate to severe COVID-19. Tears were collected within 48 hours of laboratory confirmation using 3 methods: conjunctival swab plus Schirmer's test strips (group 1), conjunctival swab (group 2), and Schirmer's test strips (group 3). Samples from both the eyes of each patient were transported in a single viral transport media for real-time RT-PCR. Detailed demographic profiles, systemic symptoms, comorbidities, and ocular manifestations were noted. Viral load of a sample was determined using cycle threshold (Ct) value of E gene. A specimen was considered to show positive results if the amplification curve for the E gene crossed the threshold line within 35 cycles and if it showed positive results on an RNA-dependent RNA polymerase or open reading frame 1b gene assay. Of the 78 patients enrolled in the study, samples from 3 patients were found to be inadequate for analysis. Thirty-six patients (48%) had moderate disease, whereas 39 patients (52%) had severe disease, with no ocular involvement in any patient. In the 75 patients, RT-PCR analysis of tears showed positive results in 18 patients (24%), and 29 of 225 samples (12.9%) showed positive results. Positive results were found in 11 (14.7%), 11 (14.7%), and 7 (9.3%) patients in groups 1, 2, and 3, respectively (P = 0.3105). Mean Ct values in groups 1, 2, and 3 were 28.36 ± 6.15, 29.00 ± 5.58, and 27.86 ± 6.46 (P = 0.92), respectively. Five patients showed positive RT-PCR results by all 3 methods (mean Ct value, 25.24 ± 6.33), and 12 patients showed positive results by any of the 3 methods (mean Ct value, 32.16 ± 1.94), the difference in Ct values being statistically significant (P = 0.029). The median value of symptomatology in patients with positive RT-PCR results from tears was 5 days (range, 4–9 days). SARS-CoV-2 RNA was detected in tears of 24% of patients with laboratory-proven moderate to severe COVID-19. Conjunctival swab remains the gold standard of tear collection for RT-PCR assay. A significantly higher possibility of viral transmission exists through tears in patients with moderate to severe COVID-19. SARS CoV-2 viral RNA was detected in tears of 24% of laboratory proven moderate to severe COVID 19 patients, in early course of the disease, posing risk of viral transmission through tears. [ABSTRACT FROM AUTHOR]

Background: Hepatitis C virus (HCV) infection, predominantly transmitted by exposure to infected blood, remains one of the major public health problems worldwide. This study aims to identify the risk factors of HCV transmission and its chronic complications among the study group., Materials and Methods: This retrospective study was approved by the Research and Ethical Review and Approve Committee (RERAC) of Oman and conducted at a secondary-care hospital situated in the North Batinah region of Oman. The study population included all HCV cases confirmed by positive serology and reverse-transcription polymerase chain reaction tests during their presence at the hospital between January 2017 and December 2022. The relevant data of the study population were retrieved from the hospital electronic health record system. The data were analyzed using the Statistical Package for the Social Sciences (SPSS), Version 26.0., Results: A total of 177 HCV confirmed cases were included in the study. HCV infection was predominant among males (74%) and individuals of the age group of 21-60 years (74.6%). Genotyping was possible only in 107 cases. Among HCV genotypes, genotype 3 (58.9%) was the most frequently identified, followed by genotype 1 (34.6%). Hemodialysis (21.5%), history of blood transfusion (16.4%), and injection drug use (11.9%) were the major risk factors for HCV infection, while cirrhosis (7.3%) and fatty liver disease (4%) were the most frequently observed chronic HCV complications. HCV infection in the spouse/partner (21.5%), alcohol use (7.3%), and co-infection with hepatitis B virus (2.3%) and human immunodeficiency virus (1.7%) were the other significant factors detected in our study population., Conclusions: HCV is a multi-factorial disease leading to severe chronic complications, thus representing a public health threat. This clearly emphasizes the cruciality of HCV community awareness campaigns and enhancement of Omani national guidelines for early screening of high-risk groups as well as effective management of HCV-infected cases to reduce the substantial burden of the disease on patients as well as the healthcare system., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Journal of Education and Health Promotion.)

Efforts towards slowing down coronavirus (COVID-19) transmission and reducing mortality have focused on timely case detection, isolation and treatment. Availability of laboratory COVID-19 testing capacity using reverse-transcriptase polymerase chain reaction (RT-PCR) was essential for case detection. Hence, it was critical to establish and expand this capacity to test for COVID-19 in Ethiopia. To this end, using a three-phrased approach, potential public and private laboratories with RT-PCR technology were assessed, capacitated with trained human resource and equipped as required. These laboratories were verified to conduct COVID-19 testing with quality assurance checks regularly conducted. Within a 10 month period, COVID-19 testing laboratories increased from zero to 65 in all regional states with the capacity to conduct 18,454 tests per day. The success of this rapid countrywide expansion of laboratory testing capacity for COVID-19 depended on some key operational implications: the strong laboratory coordination network within the country, the use of non-virologic laboratories, investment in capacity building, digitalization of the data for better information management and establishing quality assurance checks. A weak supply chain for laboratory reagents and consumables, differences in the brands of COVID-19 test kits, frequent breakdowns of the PCR machines and inadequate number of laboratory personnel following the adaption of a 24/7 work schedule were some of the challenges experienced during the process of laboratory expansion. Overall, we learn that multisectoral involvement of laboratories from non-health sectors, an effective supply chain system with an insight into the promotion of local production of laboratory supplies were critical during the laboratory expansion for COVID-19 testing. The consistent support from WHO and other implementing partners to Member States is needed in building the capacity of laboratories across different diagnostic capabilities in line with International Health Regulations. This will enable efficient adaptation to respond to future public health emergencies.

Purpose: The present study tested a recently introduced bone substitute material (BSM) with a novel structure to determine its osteoinductive and osteoconductive properties in vitro and in vivo. The specific aims were to determine the microstructure of the as-manufactured BSM, as analyzed with scanning electron microscopy, and to characterize different cellular interactions. Materials and Methods: Human bone marrow stromal cells were cultured in the presence of the BSM. In vitro, attachment of osteoblastlike cells (SAOS-2) to the BSM was observed with the scanning electron microscope. The expression of genes related to osteogenic differentiation (alkaline phosphatase, bone sialoprotein, type I collagen, and osteocalcin) was determined by reverse-transcriptase polymerase chain reaction. In vivo, bone formation was examined with a murine model of ectopic bone formation through histology and computed tomographic scanning by using tissue-engineered constructs with the BSM and ovine bone marrow stromal cells. Results: Early cellular attachment could be detected as early as 6 hours. Cellular morphology developed in the following 66 hours toward a starlike appearance. Human bone marrow stromal cells cultured in the presence of the BSM showed no reduction in their viability. Osteocalcin was up-regulated during cell culturing, demonstrating an osteoinductive effect of BSM. Histologic and computed tomographic analyses showed the formation of new bone surrounding BSM particles, and a vascular meshwork was observed in the porosity of the particles. Conclusion: The analyzed bone substitute of synthetic origin presented osteoinductive properties that may exert a differentiative stimulus upon osteoprogenitor cells. The tested material allowed cellular adhesion of osteoblastlike cells and, following tissue construct implantation in vivo, supported the formation of new bone. [ABSTRACT FROM AUTHOR]

Background: COVID-19 pandemic has stirred an unexpected turmoil in health care worldwide. The strategic conduct of critical care would warrant an effective preparedness plan, thorough knowledge of the disease manifestations, and relevant therapeutic strategy to sail through this crisis. Methods: English medical literature with MeSh database was searched using the key words such as "COVID-19 pandemic," "ICU preparedness," "COVID-19 management," "COVID-19 therapy," and "COVID-19 systemic effects." The relevant studies were included with significant inputs from interdepartmental meetings for the formulation of a plan. Results: The analysis of the respective studies highlighted the requirements of COVID-19 designated intensive care units (ICUs) with special provisions and the therapeutic agents being used in critical patients with emphasis on Remdesivir, Dexamethasone, Convalescent Plasma, and insight into newer agents. The systemic manifestations of COVID-19 requiring ICU care such as acute respiratory distress syndrome, myocardial injury, arrhythmias, hypercoagulable state, and acute renal dysfunction have been highlighted. The need of updating records with research protocols cannot be disregarded. The care of patients should not compromise the health-care personnel requirements. Conclusion: The evidence-based preparedness strategy can curtail the critical care crunch in COVID-19 management; however, institutional specific approach should be formulated. [ABSTRACT FROM AUTHOR]

Intr oducti on: The diagnosis of Ewing sarcoma family of tumours (ESFT) is challenging, especially in adults and in extra-skeletal or visceral location. Several morphologic mimics with varied treatment options and prognosis confer diagnostic dilemmas. Application of ancillary diagnostic modalities in surgical pathology in clinical routine has enabled accurate diagnosis of ESFT in bone, soft tissues, and viscera. Aim: The study aims to assess the clinicopathological features including molecular test results of ESFT with emphasis on sex, age, and location, especially extra-skeletal soft tissue and visceral location. Materia l and Met hods: Data of clinicopathological, molecular tests (wherever performed), diagnosis rendered in 302 ESFT over a decade from our centre were reviewed. Statistical comparison of skeletal and extra-skeletal tumours with reference to age and sex was done using SPSS package. The P value of <.05 was considered significant. Res ults: The cohort included 302 ESFTs with 49% skeletal and 51% extra-skeletal tumours. Thigh was most common site among skeletal tumours; chest wall, paraspinal location, and retroperitoneum among soft tissues (39.4%); and kidney, ovary, and cervix among visceral tumours (11.3%). Fluorescence in situ hybridisation for EWSR1 gene rearrangement was positive in 54 patients and reverse-transcriptase polymerase chain reaction in 19 patients. Predominance of male sex, younger age and location in extremities among skeletal tumours and lack of gender predilection, higher age and axial location in extra-skeletal tumours were noted, which were statistically significant. Molecular tests were performed more frequently in extra-skeletal tumours, especially in visceral tumours to establish the diagnosis. Conclusi ons: The study showed statistically significant differences in the age, sex, and location between skeletal and extra-skeletal ESFT. The increased percentage of extra-skeletal tumours especially in viscera was attributed to the increased awareness and availability of ancillary techniques. [ABSTRACT FROM AUTHOR]

Introduction: Acute myeloid leukemia (AML) is a clonal accumulation of myeloid precursors in body tissues, which ultimately leads to bone marrow failure. This is an 8-year prospective, observational study in which 254 patients were enrolled. Aim of the Study: To document the clinical profile of AML and differential outcome in M3 versus non-M3 phenotype and to see impact of different variables on its survival. Methods: Patients enrolled in the study were examined, evaluated, and given standard 3:7 induction protocol, and acute promyelocytic leukemia (APML) patients were given the ICAPL 2006 protocol. Results: In our study, males outnumbered females and most of our patients were in 20–60 years of age group. The better prognosis was in patients who were in the second decade of life. Total leukocyte count and platelet count had a significant impact on the survival of the a patient. Bone marrow morphology of M3 type has extremely good prognosis and was the most common FAB type seen in our study. Flow cytometric markers such as CD15, CD33, CD117, and myeloperoxidase had positivity among 90% of patients. Overall survival is around 40% in whole-study group, 87% in APML group, and 16.5% in non-M3 group. There are still unmet needs in managing the non-M3 patients in resource-constraint countries where allogenic transplant and newer drugs have the least access. For improving the outcome in M3 AML, further newer molecules such as Flt3 and PIK3 inhibitors are being used in trials. Conclusion: There are still unmet needs in managing the non-M3 patients in resource-constraint countries where allogenic transplant and newer drugs have the least access. For improving the outcome in M3 AML, further newer molecules such as Flt3 and PIK3 inhibitors are being used in trials. [ABSTRACT FROM AUTHOR]

Bovine viral diarrhea virus (BVDV) is a common pathogen that causes major economic losses to dairyindustry worldwide. Amplification of 5’-UTR regions of BVDV genome from blood samples usingreversetranscriptase polymerase chain reaction (RT-PCR) is one of sensitive and accurate techniques forBVDV detection. A common limitation for molecular detection of virus is the ability to obtain high qualityof nucleic acid in the samples. Inappropriate storage conditions will result in a false negative test. FiveEDTA-blood samples collected from BVDV persistently infected cattle were used in the present study.Ten repetitions were done for each sample. The blood were then dripped in the middle part of the FlindersTechnology AssociatesTM (FTA) cards and let it dried for approximately 2 hours. The effectiveness of theFTATMcards for storage and retrieval of BVDV RNA from blood samples molecularly using RT-PCRtechnique were tested and evaluated. The results proved that FTATM cards are effective and safe for thestorage of BVDV genome for a long period at room temperature. Based on the result of the present study,it can be concluded that in veterinary medicine field, the application of FTATM technology needs to bedeveloped to various type of samples and should be used routinely for collecting samples containinginfectious agents.

To reveal the specific features of hematopoiesis in patients with head and neck squamous cell carcinoma (HNSCC), the authors analyzed the indicators of 41 bone marrow aspirates in relation to the clinical and morphological characteristics of the tumor and micrometastases detected in bone marrow. Puncture samples were morphologically and molecularly examined at the Laboratory of Hematopoiesis Immunology, N.N. Blokhin Russian Cancer Research Center. The found changes in the bone marrow cell composition in HNSCC patients showed themselves as an increase in the relative count of segmented neutrophils and a decrease in the level of immature granulocytes with no alternations in the neutrophil maturation index and in the percentage of granulocyte lineage cells. Bone marrow hypocellularity is a characteristic feature of high-grade HNSCC. The significantly enhanced leuko-erythroblastic ratio was due to a reduction in red blood cells: the erythroid cell maturation index rose, by increasing the percentage of bone marrow oxyphilic normoblasts in patients with locally advanced HNSCC. A highly sensitive method for detecting mRNA of the specific squamous cell carcinoma protein E48 was perfected using reverse-transcription polymerase chain reaction and Southern blotting, followed by an enzyme immunoassay that could recognize bone marrow micrometastases in 35 % of the patients with HNSCC. The patients with found bone marrow micrometastases showed a significant decrease in the proportion of polychromatophilic normoblasts and a significant increase in granulocytes in the presence of normal values of lymphocytes and other cellular elements of myelograms. It was established that in the presence of regional lymph node involvement, bone marrow micrometastases were significantly more frequently in the patients with HNSCC and tumor progression over a period of up to 9 months.

Abstract Para-aminobenzoic acid (PABA) induced resistance against Pseudomonas syringae pv. tomato in Solanum lycopersicum. Fertilizer application was necessary for induced resistance and only two out of eight breeding lines showed PABA-induced resistance. A comparison of gene expression between a PABA-responsive and PABA-non-responsive line showed that either the responsive line had a faster and stronger increase in expression or the responsive line showed increased expression due to PABA. Although PABA affected SA-related gene expression, changes in gene expression were not always directly related to the responsiveness to PABA. The response to PABA by SA, JA and ET-related mutants showed SA dependence. Factors, such as fertilization practices and host genotype, are important considerations in the development of PABA as a disease management product. Highlights • PABA induced resistance to P. syringae pv. tomato in S. lycopersicum. • S. lycopersicum response to PABA was dependent on fertilization and genotype. • PABA induced SA-related defense gene expression pre and/or post-inoculation. • Breeding lines differed in the patterns of defense gene expression. • Response of SA, JA, and ET mutants to PABA confirmed a SA dependent response. [ABSTRACT FROM AUTHOR]

The aim of the present investigation was to describe a new model of liver regeneration in growing rats with reduced portal flow. In addition, it was studied whether tacrolimus and insulin could have any pro-regenerative effect under such conditions. Ninety-five rats were divided into five groups: Group 1 (sham), abdominal incision without intervention; Group 2, 70 % hepatectomy; Group 3, 70 % hepatectomy + PV stenosis; Group 4, 70 % hepatectomy + portal vein stenosis + insulin; and Group 5, 70 % hepatectomy + portal vein stenosis + tacrolimus. The remnant liver lobes were harvested for analyses. The liver weight decreased in the PV stenosis group and it increased with the use of insulin and tacrolimus. The mitotic activity was higher in the hepatectomy, insulin and tacrolimus groups and this parameter was reduced by portal stenosis. Levels of interleukin 6 (IL-6) were higher in the hepatectomy group compared to the sham and PV stenosis groups. The expression of IL-6 and Ki67 was significantly increased in the insulin and tacrolimus groups compared to the portal stenosis group. A highly reproducible model was standardized to study liver regeneration with portal blood inflow reduction in weaning rats. It was demonstrated that insulin or tacrolimus administration may partially reverse the harmful effects of PV stenosis. [ABSTRACT FROM AUTHOR]

Patient: 33-year-old male. Chief Complaint: Intermittent fevers, joint pain, gross hematuria Past Medical History: Eight years status post orthotopic liver transplant for hepatitis B virus and hepatocellular carcinoma. Allograft is stable with no evidence of rejection. Medications: Prograf and tenofovir Physical Exam Findings: Alert, oriented, thin gentleman, no thrush. Family History: Adopted, unknown Social History: Recent travel to Sudan and Kenya for 3 weeks on a hunting trip; returned 3 weeks prior to admission. The patient reports several mosquito exposures and ate buffalo, which he skinned and cooked on the riverbank. He walked through flood waters from the Nile River but did not swim in any lakes or rivers. Principal Laboratory Findings: See Table 1 [ABSTRACT FROM AUTHOR]

Purpose: The present study tested a recently introduced bone substitute material (BSM) with a novel structure to determine its osteoinductive and osteoconductive properties in vitro and in vivo. The specific aims were to determine the microstructure of the as-manufactured BSM, as analyzed with scanning electron microscopy, and to characterize different cellular interactions. Materials and Methods: Human bone marrow stromal cells were cultured in the presence of the BSM. In vitro, attachment of osteoblastlike cells (SAOS-2) to the BSM was observed with the scanning electron microscope. The expression of genes related to osteogenic differentiation (alkaline phosphatase, bone sialoprotein, type I collagen, and osteocalcin) was determined by reverse-transcriptase polymerase chain reaction. In vivo, bone formation was examined with a murine model of ectopic bone formation through histology and computed tomographic scanning by using tissue-engineered constructs with the BSM and ovine bone marrow stromal cells. Results: Early cellular attachment could be detected as early as 6 hours. Cellular morphology developed in the following 66 hours toward a starlike appearance. Human bone marrow stromal cells cultured in the presence of the BSM showed no reduction in their viability. Osteocalcin was up-regulated during cell culturing, demonstrating an osteoinductive effect of BSM. Histologic and computed tomographic analyses showed the formation of new bone surrounding BSM particles, and a vascular meshwork was observed in the porosity of the particles. Conclusion: The analyzed bone substitute of synthetic origin presented osteoinductive properties that may exert a differentiative stimulus upon osteoprogenitor cells. The tested material allowed cellular adhesion of osteoblastlike cells and, following tissue construct implantation in vivo, supported the formation of new bone. [ABSTRACT FROM AUTHOR]

Abstract: Full-length genome sequencing of the rabies virus is not a routine laboratory procedure. To understand fully the epidemiology, genetic variation and evolution of the rabies virus, full-length viral genomes need to be obtained. For rabies virus studies, cDNA synthesis is usually performed using nonspecific oligonucleotides followed by cloning. When specific primers are used, the cDNA obtained is only partial and is limited to the coding regions. Therefore, the development of methods for synthesizing long cDNA using rabies virus-specific primers is of fundamental importance. A new protocol for the synthesis of long cDNA and the development of 19 new primers are described in this study. This procedure allowed the efficient amplification of the full-length genome of the rabies virus variant maintained by hematophagous bat (Desmodus rotundus) populations following the synthesis of a complete long cDNA. Partial sequencing of the rabies virus genome was performed to confirm rabies-specific PCR amplification. Because degenerate primers were employed, this technique can be adapted easily to other variants. Importantly, this new method is faster and less expensive than cloning methods. [Copyright &y& Elsevier]

Abstract: Unprecedented biotechnological advances in the past decade have delivered powerful transcriptomics methods that provide new opportunities for a risk-based and, hence, more effective control of food quality and safety. The fundamental hypothesis underlying the application of a transcriptomics or other “-omics” strategies is that toxicant-specific changes of gene expression will reveal the presence of chemical hazards with possibly adverse health effects. Indeed, living cells display wide adaptive plasticity by responding with characteristic expression signatures to various stimuli (e.g., proliferation signals, genotoxic or cytotoxic insults, oxidative bursts and other types of chemical stress). Flexible transcriptomics platforms that can be scaled to suit different applications in food quality and safety involve oligonucleotide microarrays (also known as “DNA chips”), focused polymerase chain reaction (PCR) arrays and, more recently, high-throughput “next generation” sequencing technologies. In this review, we illustrate the potential impact of transcriptomics for the detection of hazardous food constituents or contaminants, including dioxins, xenoestrogens, mycotoxins and Maillard reaction products. We also discuss the predictive power of “-omics” fingerprinting with respect to the identification of emerging contaminants and the significance of these methods for risk assessment at all stages of the food-production chain. [Copyright &y& Elsevier]

Abstract: Introduction: This study identified the bacterial taxa enduring endodontic treatment procedures by using a combined 16S ribosomal RNA-based reverse-transcriptase polymerase chain reaction (RT-PCR) and reverse-capture checkerboard hybridization approach. Methods: Samples were taken from infected canals of 15 teeth with apical periodontitis before treatment (S1), after chemomechanical preparation with NaOCl as the irrigant (S2), and after interappointment medication with a calcium hydroxide paste (S3). Bacterial presence was first screened by a DNA-based single PCR assay. RNA extracts were subjected to RT-PCR, and the resulting products were surveyed for the presence of 28 targeted taxa by using the checkerboard method. Results: Bacteria were found in all S1 samples. Detectable levels of bacterial ribosomal RNA, used as an indicator of viability, were observed in 60% of the cases after chemomechanical preparation and 53% after intracanal medication. The most prevalent taxa in S1 were Olsenella uli (67%), Pyramidobacter piscolens (60%), Streptococcus species (53%), and Bacteroidetes clone X083 (53%). Streptococcus species (47%), Fusobacterium nucleatum (40%), and O. uli (33%) prevailed in S2, whereas Streptococcus species (47%), Propionibacterium acnes (27%), and O. uli (27%) were the most frequent taxa in S3. Conclusions: The present study with a combined molecular approach revealed that bacterial diversity was overall markedly reduced by treatment procedures. Although bacterial taxa more frequently identified in post-treatment samples emerge as potential risk factors for persistent disease, this remains to be determined by longitudinal studies. [Copyright &y& Elsevier]

Using computational methods of database analysis, we identified nucleotide sequences expressed predominantly in human tumors. Experimental evidence was obtained for one of these sequences, belonging to UniGene cluster Hs.633957 and located near the telomere of chromosome 7p22.3. The RNA sequences encoded by Hs.633957 encode no proteins, and their role in the human body is unknown. The expression pattern of the locus arouses fundamental and practical interest. We studied the expression of the Hs.633957 nucleotide sequence in normal and tumor human tissues by RT-PCR. In normal tissues of an adult human, a relatively low expression rate of this locus was observed only in heart and liver. In an embryo, expression was found in brain and kidneys. The Hs.633957 locus is expressed in tumors of various organs, including lung, intestines, breast, stomach, cervix, lymph nodes, and others. Thus, Hs.633957 is expressed predominantly in tumors and therefore may be considered a promising tumor marker. [ABSTRACT FROM AUTHOR]

Abstract: Recent evidence suggests that retinopathy of prematurity, a potentially blinding condition of premature human neonates, has a genetically-determined component. Different inbred strains of rat exhibit differential susceptibility to oxygen-induced retinopathy (OIR), a well-established experimental model of retinopathy of prematurity. To explore the basis for this differential susceptibility, we quantified the retinal expression of 8 angiogenesis-related genes during early post-natal retinal development in rats with OIR. Inbred Fischer 344 (F344), Dark Agouti (DA) and Sprague Dawley (SPD) rat neonates were exposed to alternating cycles of 80% oxygen in air and normoxia for up to 14 days. After 14 days of cyclic hyperoxic exposure, some rats were exposed to normoxia for a further 4 days. Retinal mRNA for vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), pigment epithelium-derived factor (PEDF), angiopoietin-2 (Ang2), Tie2, cyclooxygenase-2 (COX2), insulin-like growth factor-1 (IGF1) and erythropoietin (EPO) were quantified by real-time reverse-transcriptase polymerase chain reaction at different time-points. Time-course analysis showed that expression of mRNA for VEGF, VEGFR2 and Ang2 was significantly greater in OIR-resistant (F344) retinae than in OIR-susceptible (DA) retinae during the first 9 days of cyclic hyperoxia. However, at post-natal days 14 and 18, retinal mRNAs for VEGF, EPO, VEGFR2, Ang2, IGF1, COX2 and PEDF were expressed to a significantly greater extent in OIR-susceptible (DA, SPD) than OIR-resistant (F344) retinae. The VEGF/PEDF ratio was greater in the F344 compared with the DA strain up to day 9, but was higher in the DA than the F344 strain at days 14 and 18. Thus, we found that retinal expression of angiogenesis-related genes was significantly higher in OIR-resistant rats than in OIR-susceptible rats during early retinal development, but the pattern reversed during the proliferative phase of OIR. We conclude that susceptibility to OIR correlates with differential gene expression very early in retinal microvascular development, during periods of cyclic hyperoxic exposure rather than during subsequent sustained hypoxia. [Copyright &y& Elsevier]

In 2004, a virus infection of tobacco with a so far unknown virus was observed in three Central European countries, Poland, Germany and Hungary. By means of cloning and sequencing of a partial cDNA of the virus the causal agent was identified as Colombian Datura virus (CDV). Comparison of the sequences of cloned cDNAs of isolates from all three locations together with phylogenetical analysis revealed that they were closely related to each other and already published isolates, but of different origin. Polyclonal antibodies were produced which reacted specifically with CDV. They were used for immunocapture reverse-transcriptase polymerase chain reaction. In future they can be used for the virus survey in tobacco. [ABSTRACT FROM AUTHOR]

Abstract: To differentiate severe acute respiratory syndrome (SARS) from non-SARS illness, we retrospectively compared 53 patients with probable SARS and 31 patients with non-SARS who were admitted to Mackay Memorial Hospital from April 27 to June 16, 2003. Fever (> 38°C) was the earliest symptom (50/53 SARS vs. 5/31 non-SARS, p &#60; 0.0001), preceding cough by a mean of 4.5 days. The initial chest X-ray study was normal in 22/53 SARS cases versus 5/31 non-SARS cases. SARS patients with an initially normal chest X-ray study developed infiltrates at a mean of 5 ± 3.44 days after onset of fever (21/22 SARS vs. 0/5 non-SARS). Rapid radiographic progression of unifocal involvement to multifocal infiltrates was seen in 22 of 24 SARS vs. 0 of 26 non-SARS patients (p &#60; 0.0001). Pleural effusion was not present in any SARS patients but was seen in 6 of 26 non-SARS cases (p &#60; 0.0001). Initial lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase were all more common in SARS than non-SARS (p &#60; 0.0001). They may help differentiate SARS from non-SARS if a reliable and rapid diagnostic test is not available. [Copyright &y& Elsevier]

The expression of transcripts for anti-apoptotic (survivin, survivin-ΔEx3, survivin-2B, galectin-3, bag-1 and bcl-2) and pro-apoptotic (bax-α) genes, and for multiple drug resistance related protein-1 (MRP-1) gene were investigated, using RT-PCR, in 106 breast tumour biopsies. Normal breast tissue was also analysed for comparative purposes. Overall, survivin, survivin-ΔEx3, survivin-2B, bcl-2, bag-1, galectin-3, bax-α and MRP-1 mRNAs were detected in 68, 54.7, 9.4, 78.4, 80.9, 98.9, 97.8 and 72.8%, respectively, of tumour specimens. Uniquely among the mRNAs analysed, the expression of bcl-2 correlated significantly with disease outcome, with bcl-2 expression indicative of favourable outcome in terms of both relapse-free survival and overall survival. This suggests that bcl-2 mRNA expression may be a key prognostic marker for breast cancer and that routine analysis of expression of this transcript should be considered. The results from this study suggest, however, that the expression of survivin, survivin-ΔEx3, survivin-2B, bag-1, galectin-3, bax-α and MRP-1 mRNAs cannot be considered as prognostic indicators of disease outcome for patients with breast cancer. [Copyright &y& Elsevier]

Aim: To determine the expression of two isoforms of the growth hormone (GH) receptor (GHR), which differ by the presence (GHR3+) or absence (GHR3–) of exon 3, and their correlation with circulating GH and insulin-like growth factor I (IGF-I) in normal subjects and in acromegalic patients. Methods: The expression of GHR isoforms was determined by reverse-transcriptase polymerase chain reaction in lymphocytes from 12 normal subjects and from 11 patients with acromegaly. The levels of GHR mRNA were normalized to those of β-actin, and ratios were calculated to assess the relative levels of expression. Results: All samples showed expression of both GHR isoforms, but the expression of GHR3+ and GHR3– was similar in acromegalic patients (6.0 ± 1.7 vs. 8.3 ± 2.0%, mean ± SE). In contrast, in healthy subjects, GHR3– was the predominant isoform (11.8 ± 3.0 vs. 5.1 ± 0.68%; p < 0.05), and the levels of expression of GHR3– correlated significantly with IGF-I. Conclusions: These data demonstrate coexpression of both GHR isoforms under normal and pathological conditions; however, GHR3– is the predominant form in normal subjects and shows a negative correlation with IGF-I levels.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Recently, a species-dependent distribution of melatonin binding sites have been found in lamina I–V and lamina X of the spinal cord. In order to learn more about the function of spinal melatonin receptors, we investigated (i) the gene expression for melatonin receptor subtypes in lumbar and thoracal spinal cord tissue by means of the reverse-transcriptase polymerase chain reaction (RT-PCR) technique, and (ii) the electrophysiological and pharmacological properties of melatonin receptors heterologously expressed in Xenopus oocytes after injection of spinal cord mRNA by means of the voltage clamp technique. Because ample evidence indicates an antinociceptive effect of melatonin, (iii) the role of spinal melatonin receptors for maintaining mechanical and thermal hyperalgesia was studied in a rat model for postoperative pain. The RT-PCR data revealed that transcripts for MT1 and MT2 melatonin receptors are present in the dorsal and ventral horn of lumbar and thoracal spinal cord tissue. Injection of mRNA from lumbar spinal cord tissue into Xenopus oocytes led to the functional reconstitution of melatonin receptors which activate calcium-dependent chloride inward currents. Melatonin responses were abolished by simultaneous administration of the antagonists, 2-phenylmelatonin and luzindole and were unaffected by the MT2 antagonist 4-phenyl-2-propionamidotetralin. Intrathecal administration of different melatonin doses (10–100 nmol) did not inhibit mechanical or thermal hyperalgesia. However, intrathecal application of a low dose of morphine together with melatonin caused a brief antinociceptive effect suggesting an enhanced morphine analgesia by melatonin. In conclusion, the present study demonstrated for the first time the presence of transcripts of MT1 and MT2 receptors located in the dorsal and ventral horn of the spinal cord. Furthermore, spinal melatonin enhanced the antinociceptive effect of... [ABSTRACT FROM AUTHOR]

Drosophila melanogaster has a gene very similar to human MRP1 that encodes a full ABC-transporter containing three membrane-spanning domains and two nucleotide-binding domains. This 19 exon insect gene, dMRP (FBgn0032456), spans slightly more than 22 kb. The cDNA SD07655 representing this gene was sequenced and found to contain sequences from 12 exons including single copies of two exons having multiple genomic copies. The gene contains two variant copies of exon 4 and seven of exon 8. While a cDNA contains only one version of each variable exon, all forms of these variable exons were detected in adult fly mRNA. These results predict that Drosophila could make 14 different MRP isoforms from a single gene by substituting different variable exons. This is the first report of any organism using differential splicing of alternative, internal exons, to produce such a large array of MRP isoforms having the same size, but with limited and defined internal variations. Defining the functional differences in the dMRP isoforms should provide clues to the structure/function relationships of the amino acids in these MRP domains, both for the insect enzyme and for those of other species. [Copyright &y& Elsevier]

: ObjectivesWe sought to study the expression of bradykinin type-2 receptors (BK-2Rs) in patients with heart failure (HF).: BackgroundRecent work in experimental animals has suggested that bradykinin (BK) exerts cardioprotective effects through specific BK-2Rs. However, nothing is known about the regulation of BK-2R expression in the pathogenesis of human HF.: MethodsHuman heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13) and from normal hearts (n = 6) unsuitable for donation. The patients had HF due to idiopathic dilated cardiomyopathy (IDC) (n = 7) or coronary heart disease (CHD) (n = 6). Tissue samples from the left ventricles were analyzed by competitive reverse-transcriptase-polymerase chain reaction and Western blotting for the expression of BK-2R messenger ribonucleic acid (mRNA) and protein.: ResultsIn both the IDC and CHD hearts, the level of BK-2R mRNA expression was found to be significantly lower (30% and 38% of control values, respectively) than that in normal hearts. Correspondingly, the BK-2R protein level was significantly reduced in both the IDC and CHD hearts (45% and 62% of control values, respectively) and apparently involved all myocardial cell types. The down-regulation of BK-2R expression in failing hearts did not correlate with decreased cellularity or with the expression pattern of other members of the G-protein–coupled receptor superfamily. However, BK-2R down-regulation in the failing hearts was associated with a decrease in endothelial nitric oxide synthase in both IDC (53% of control value) and CHD (43% of control value) hearts.: ConclusionsThese results are the first to suggest that a loss of BK-2Rs is involved in the pathogenesis of human HF. [Copyright &y& Elsevier]

For patients with chronic myeloid leukaemia, methods for monitoring response to treatment have changed considerably in recent years. In the 1980s, the principal approach was repeated examination of bone marrow metaphases for the presence of the Ph chromosome in patients treated by interferon-α (IFN-α) or allogeneic stem cell transplantation. The use of fluorescence in situ hybridisation (FISH) techniques to detect the BCR-ABL fusion gene in Ph-positive leukaemia cells increased the sensitivity of cytogenetic studies to some degree. In the last 10 years, the reverse-transcriptase polymerase chain reaction (RT-PCR) has proved extremely valuable for assessing and monitoring minimal residual disease in patients who achieve Ph negativity after treatment with IFN-α or with the new Abl tyrosine kinase inhibitor imatinib mesylate or after allogeneic stem cell transplantation (SCT). Results are consistent with the notion that the majority of long-term survivors after allogeneic SCT are probably ‘cured’; for other patients monitored serially in complete cytogenetic remission, rising numbers of BCR-ABL transcripts detected by RT-PCR can indicate the need for further therapy.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

To examine whether cancer cell dissemination results from incisional biopsy, we tried to detect squamous cell carcinoma (SCC) cells in peripheral blood before and after incisional biopsy by means of cytokeratin 19 (CK19) reverse-transcriptase polymerase chain reaction (RT-PCR). The study population consisted of 20 patients with oral SCC; 10 were given incisional biopsies followed by radical excision (the incisional biopsy group), and the remaining 10 were treated by excisional biopsy alone (the excisional biopsy group). Ten non-oral cancer patients with benign oral lesions served as controls. Five-ml blood aspirates collected before and after incision were used for CK19 RT-PCR. Two (20.0%) of 10 patients from the incisional biopsy group were positive for CK19 transcripts in their peripheral blood drained 15 min after incision. In contrast, CK19 transcript was not detected either in the excisional biopsy group or in controls. Surgical invasiveness for oral cancer, including incisional biopsy, causes dissemination of cancer cells into circulation, resulting in increased risk of metastasis. [ABSTRACT FROM AUTHOR]

We have used RT-PCR and GFP-mediated fluorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells. Our data show that most of the PrfA-regulated virulence genes are more efficiently expressed, as measured by transcript levels, when L. monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells. The promoters for hly and plcA are predominantly activated within the phagosomal compartment, while those for actA and inlC are predominantly activated in the host cell cytosol. Expression of actA and plcB precedes that of inlC after infection of epithelial cells and macrophages. Little transcription of inlA or inlB is observed in epithelial cells and there is only slightly more in macrophages. In both cell types the level of transcription of the inlAB operon is lower than is seen under extracellular growth conditions in rich media, which is compatible with the assumption that InlA and InlB are not required during intracellular growth of the bacteria. Activation of the PrfA-independent iap promoter is also low during intracellular growth, although the gene product (p60) is required for cell viability. The levels of the PrfA-dependent virulence gene transcripts do not correlate with the amount of prfA transcript present, which is low under all intracellular conditions analysed, suggesting that the prfA transcript is either highly unstable in bacteria that are growing intracellularly, or that the small amount of PrfA produced is highly activated by additional component(s). [ABSTRACT FROM AUTHOR]

Analysis of the variable chains (Vα/Vβ) of the specific T cell receptor (TCR) of organ-infiltrating T cells may provide further insights into the pathogenesis of many infectious diseases, malignancies, and autoimmune disorders. To determine the TCR Vβ repertoire of these small T cell populations antigen-independent in vitro expansion is necessary but may select for certain T cell subpopulations. In this study various antigen independent T cell activation protocols were used to stimulate peripheral blood mononuclear cells (PBMC) of six healthy blood donors, and TCR Vβ molecules were analyzed by flow cytometry and semiquantitative reverse-transcriptase polymerase chain reaction. In addition, the analysis of in vitro expanded liver-infiltrating T cells and autologous peripheral blood T cells derived from five patients with autoimmune hepatitis but none of six controls revealed a selective overexpression of single TCR Vβ molecules in the liver tissue. In contrast to freshly isolated PBMC, no preferential expansion of single TCR Vβ families was observed using phytohemagglutinin, anti-CD3 antibodies, or oxidative stress for antigen-independent T cell activation. In conclusion, antigen-independent T cell activation offers the chance to analyze small populations of organ-infiltrating T cells without skewing the TCR Vβ repertoire. [ABSTRACT FROM AUTHOR]

Efforts towards slowing down coronavirus (COVID-19) transmission and reducing mortality have focused on timely case detection, isolation and treatment. Availability of laboratory COVID-19 testing capacity using reverse-transcriptase polymerase chain reaction (RT-PCR) was essential for case detection. Hence, it was critical to establish and expand this capacity to test for COVID-19 in Ethiopia. To this end, using a three-phrased approach, potential public and private laboratories with RT-PCR technology were assessed, capacitated with trained human resource and equipped as required. These laboratories were verified to conduct COVID-19 testing with quality assurance checks regularly conducted. Within a 10-month period, COVID-19 testing laboratories increased from zero to 65 in all Regional States with the capacity to conduct 18,454 tests per day. The success of this rapid countrywide expansion of laboratory testing capacity for COVID-19 depended on some key operational implications: the strong laboratory coordination network within the country, the use of non-virologic laboratories, investment in capacity building, digitalization of the data for better information management and establishing quality assurance checks. A weak supply chain for laboratory reagents and consumables, differences in the brands of COVID-19 test kits, frequent breakdowns of the PCR machines and inadequate number of laboratory personnel following the adaption of a 24/7 work schedule were some of the challenges experienced during the process of laboratory expansion. Overall, we learn that multisectoral involvement of laboratories from non-health sectors, an effective supply chain system with an insight into the promotion of local production of laboratory supplies were critical during the laboratory expansion for COVID-19 testing. The consistent support from WHO and other implementing partners to Member States is needed in building the capacity of laboratories across different diagnostic capabilities in line with International Health Regulations. This will enable efficient adaptation to respond to future public health emergencies., Competing Interests: The authors declare no competing interests., (Copyright: Adisu Kebede et al.)

In situ hybridization is an ideal tool for the detection and localization of mRNA expression of specific gene(s) in tissue sections and cell lines for prognosis, predictive markers, and highlighted potential therapeutic targets. Given the importance of CCN1 and CCN5 in breast and pancreatic cancer progression, these two secretory proteins could be novel therapeutic targets. Thus, evaluating the distribution of mRNA of these targets using in situ hybridization could be important preclinical tools. This chapter describes a detailed in situ hybridization technique for the detection of CCN1 and CCN5 in formalin-fixed, paraffin-embedded patient samples of breast and pancreatic cancers.

Background: Although systemic hypertension is closely associated with aortic aneurysm (AA) formation, there are many patients with AA without hypertension. In these patients, an inflammation-mediated progression of aneurysmal disease is likely responsible for AA growth and eventual rupture. Unfortunately, there remains no reproducible and durable small animal model of aortic aneurysmal disease, the development of which would enable the investigation of the pathophysiology of this vexing condition. The first aim was to establish a useful wild-type mouse model of AA with low mortality. The second aim was to use this model to assess the protective effect of azelnidipine, a new calcium channel blocker, against the progression of the AA independent of its antihypertensive effect., Methods: Angiotensin II and β-aminopropionitrile (a lysyl oxidase inhibitor) were administrated subcutaneously in 7-week-old C57BL/6J mice using an osmotic minipump for 4 weeks to generate a wild-type mouse model of AA. Concurrently, azelnidipine (a calcium channel blocker) or a placebo was administrated orally for 4 weeks. Mice were humanely killed and assessed at the end of the 4 weeks of pharmacologic manipulation., Results: The combined infusion of angiotensin II and β-aminopropionitrile induced degenerative aneurysm of the thoracic and/or abdominal aorta (11/12; 92%). The majority of aneurysms were located in the distal aortic arch and suprarenal abdominal aorta. Although there was no difference in systolic blood pressure between the control and azelnidipine-treated groups, azelnidipine significantly reduced the incidence of AA (2/11; 18%). Azelnidipine treatment reduced the pathologic findings normally associated with aneurysm formation within the aortic wall. Azelnidipine also reduced the number of macrophage antigen-3 (MAC-3)-positive cells in the periaortic adipose tissue and reduced the gene expression levels of tumor necrosis factor-alpha and matrix metalloproteinase-2 and -9 within the aortic wall., Conclusions: This study demonstrates that combined treatment with angiotensin II and β-aminopropionitrile induces degenerative AAs in wild-type mice, and azelnidipine prevents aneurysm progression via its anti-inflammatory effect., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Background: Human metapneumovirus (HMPV) is a recently discovered paramyxovirus that is a major cause of respiratory infections worldwide., Objectives: We aim to describe the molecular evolution of the HMPV F (fusion) and G (attachment) surface glycoproteins because they are targets for vaccines, monoclonal antibodies and antivirals currently in development., Study Setting: Nasopharyngeal aspirates were collected in children <3 years old with acute respiratory infection in Quebec City during 2001-2010. HMPV-positive samples (n = 163) underwent HMPV-F and -G gene sequencing. Furthermore, HMPV-F (n = 124) and -G (n = 217) sequences were obtained from GenBank and other studies. Evolutionary analyses (phylogenetic reconstruction, sequence identity, detection of recombination and adaptive evolution) were computed., Results: Sequences clustered into 5 genetic lineages (A1, A2a, A2b, B1 and B2). Multiple lineages circulated each year in Quebec City. With the exception of B1, each of the 5 subgroups was the predominant lineage during ≥1 season. The A1 lineage was not detected since 2002-2003 in our local cohort. There was no evidence of inter- or intragenic recombination. HMPV-F was highly conserved, whereas HMPV-G exhibited greater diversity. HMPV-F demonstrated strong evidence of purifying selection, both overall and in an abundance of negatively selected amino acid sites. In contrast, sites under diversifying selection were detected in all HMPV-G lineages (range, 4-15), all of which were located in the ectodomain., Conclusions: Predominant circulating HMPV lineages vary by year. HMPV-F is highly constrained and undergoes significant purifying selection. Given its high genetic variability, we found a modest number of positively selected sites in HMPV-G., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Respiratory syncytial virus rapid antigen detection tests (RADT) are used widely. RADT exhibited high specificity (97%) and moderate sensitivity (80%) compared with reverse-transcriptase polymerase chain reaction in 720 hospitalized children <3 years old. Older age, prolonged symptoms, and respiratory syncytial virus genotype-B infection were significantly associated with false-negative results of RADT., (Copyright © 2013 Mosby, Inc. All rights reserved.)