A cis-regulatory sequence of the selector gene vestigial drives the evolution of wing scaling in Drosophila species.

Scaling between specific organs and overall body size has long fascinated biologists, being a primary mechanism by which organ shapes evolve. Yet, the genetic mechanisms that underlie the evolution of scaling relationships remain elusive. Here, we compared wing and fore tibia lengths (the latter as a proxy of body size) in Drosophila melanogaster, Drosophila simulans, Drosophila ananassae and Drosophila virilis, and show that the first three of these species have roughly a similar wing-to-tibia scaling behavior. In contrast, D. virilis exhibits much smaller wings relative to their body size compared with the other species and this is reflected in the intercept of the wing-to-tibia allometry. We then asked whether the evolution of this relationship could be explained by changes in a specific cis-regulatory region or enhancer that drives expression of the wing selector gene, vestigial (vg), whose function is broadly conserved in insects and contributes to wing size. To test this hypothesis directly, we used CRISPR/Cas9 to replace the DNA sequence of the predicted Quadrant Enhancer (vgQE) from D. virilis for the corresponding vgQE sequence in the genome of D. melanogaster. Strikingly, we discovered that D. melanogaster flies carrying the D. virilis vgQE sequence have wings that are significantly smaller with respect to controls, partially shifting the intercept of the wing-to-tibia scaling relationship towards that observed in D. virilis. We conclude that a single cis-regulatory element in D. virilis contributes to constraining wing size in this species, supporting the hypothesis that scaling could evolve through genetic variations in cis-regulatory elements.

Predominance of CXCR4 tropism in HIV-1 CRF14_BG strains from newly diagnosed infections.

OBJECTIVES: R5-tropic viruses are associated with HIV-1 transmission and predominate during the early stages of infection. X4-tropic populations have been detected in ~50% of patients with late-stage disease infected with subtype B viruses. In this study, we compared the frequency of X4 tropism in individuals infected with HIV-1 CRF14_BG viruses, which have a V3 loop of subtype B, with a control group of individuals infected with subtype B viruses. METHODS: Sixty-three individuals infected with HIV-1 CRF14_BG (n = 31) or subtype B (n = 32) were studied. Similar proportions of newly diagnosed and chronically infected individuals were included in the subtype B and CRF14_BG groups. V3 sequences were obtained and coreceptor tropism was predicted using the Geno2pheno[coreceptor] algorithm. V3 net charge and 11/25 rules were also used for coreceptor prediction. RESULTS: Overall, X4 tropism was more frequent among individuals infected with CRF14_BG viruses (87.1%) than subtype B viruses (34.3%), a difference that was statistically highly significant (P = 0.00001). Importantly, the frequencies among newly diagnosed individuals were 90% and 13.3%, respectively (P = 0.0007). Characteristic amino acids in the V3 loop (T13, M14, V19 and W20) were identified at higher frequencies in CRF14_BG viruses (54%) than subtype B viruses (0%; P < 0.000001). CONCLUSIONS: CRF14_BG is the genetic form with the highest proportion of X4-tropic viruses reported to date in newly diagnosed and chronic infections. This suggests high pathogenicity for CRF14_BG viruses, potentially leading to rapid disease progression. CCR5 antagonists will be ineffective in most CRF14_BG-infected patients, even at early stages of infection.

Evaluation of genotypic tropism prediction tests compared with in vitro co-receptor usage in HIV-1 primary isolates of diverse subtypes.

OBJECTIVES: To evaluate the sensitivity and specificity of genotypic methods for predicting the co-receptor usage of subtypes B and non-B HIV-1 primary isolates, using as gold standard the infectivity of each primary isolate in GHOST cells stably expressing HIV-1 co-receptors. METHODS: Primary isolates were obtained by co-culturing either patient's peripheral blood mononuclear cells (PBMCs) or ultracentrifuged plasma with donor-activated PBMCs. In vitro co-receptor usage was determined by infecting GHOST cells. Tropism prediction, based on V3 sequences, was determined with simple rules and bioinformatic tools (Geno2pheno[coreceptor] and WebPSSM). RESULTS: This study includes 102 HIV-1 primary isolates; 23 (22.5%) subtype B and 79 (77.5%) non-B genetic forms. V3 sequences were classified into six subtypes (A-G), although 32 (31.4%) were circulating recombinant forms and 21 (20.6%) were unique recombinant forms. Sixty-nine isolates were R5, 27 R5X4 and 6 X4. The highest levels of sensitivity and specificity for the detection of X4 strains among V3 sequences, between 91% and 100%, were obtained by using PSSM(x4r5), PSSM(si/nsi) and the 11/25 rule for sequences of subtypes A, B and G, but not for subtype F. Establishing the recommended cut-off for clinical settings of a 10% false positive rate for Geno2pheno, we obtained 93% specificity and 97% sensitivity. CONCLUSIONS: Comparing genotypic assays for HIV-1 co-receptor use with a cell-culture phenotypic assay could provide more reliable results of sensitivity and specificity for the detection of X4 strains than comparing them with recombinant assays, considered as gold standard. In general, except for subtype F isolates, there is a good correlation for tropism prediction.

The vestigial Quadrant Enhancer is dispensable for pattern formation and development of the Drosophila wing.

In Drosophila , the pattern of the wing selector gene, vestigial ( vg ), is established by at least two enhancers: the Boundary Enhancer, which drives expression along the disc's Dorsal-Ventral boundary; and the Quadrant Enhancer (QE) that patterns the rest of the wing pouch. Using CRISPR/Cas9 editing, we deleted DNA fragments around the reported QE sequence and found that the full Vg pattern is formed. Furthermore, adult wings arising from these gene-edited animals are normal in shape and pattern, but slightly smaller in size, although this reduction is not wing-specific in males. We suggest that other enhancers act redundantly to establish the vg pattern and rescue wing development.

Implementation of a Multiplex rRT-PCR for Zika, Chikungunya, and Dengue Viruses: Improving Arboviral Detection in an Endemic Region.

Arboviral diagnosis has been complicated throughout the tropical and subtropical Americas by the recent co-circulation of Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV). The aim of this study was to implement a multiplex real-time RT-PCR (rRT-PCR) for ZIKV, CHIKV, and DENV in Paraguay to test patients who were clinically suspected of having dengue. We tested 110 sera from patients who presented to the Hospital de Clínicas in 2016 and had testing for DENV nonstructural protein 1 (NS1; 40 positive and 70 negative). Using a composite reference standard, we confirmed 51 dengue cases (46.4%): 38/40 NS1 positive and 13/70 NS1 negative. Chikungunya virus and ZIKV were detected in one sample each, both were DENV NS1 negative. The NS1 test demonstrated good agreement with rRT-PCR for DENV. However, multiplex rRT-PCR identified a subset of dengue cases and additional arboviral infections that would not be detected if NS1 assays are relied upon for diagnosis.

Fish allergy in childhood.

Fish and its derived products play an important role in human nutrition, but they may also be a potent food allergen. Fish can be an ingested, contact, and inhalant allergen. Gad c I, a Parvalbumin, the major allergen in codfish, is considered as fish and amphibian pan-allergen. Prevalence of fish allergy appears to depend on the amount of fish eaten in the local diet. In Europe, the highest consumption occurs in Scandinavian countries, Spain and Portugal. In Spain, fish is the third most frequent allergen in children under 2 yr of age after egg and cow's milk. An adverse reaction to fish may be of non-allergic origin, due to food contamination or newly formed toxic products, but the most frequent type of adverse reactions to fish are immunologic-mediated reactions (allergic reactions). Such allergic reactions may be both IgE-mediated and non-IgE-mediated. Most cases are IgE-mediated, due to ingestion or contact with fish or as a result of inhalation of cooking vapors. Some children develop non-IgE-mediated type allergies such as food protein induced enterocolitis syndrome. The clinical symptoms related to IgE-mediated fish allergy are most frequently acute urticaria and angioedema as well as mild oral symptoms, worsening of atopic dermatitis, respiratory symptoms such as rhinitis or asthma, and gastrointestinal symptoms such as nausea and vomiting. Anaphylaxis may also occur. Among all the species studied, those from the Tunidae and Xiphiidae families appear to be the least allergenic.

Krüppel-Like Factor 10 participates in cervical cancer immunoediting through transcriptional regulation of Pregnancy-Specific Beta-1 Glycoproteins.

Cervical cancer (CC) is associated with alterations in immune system balance, which is primarily due to a shift from Th1 to Th2 and the unbalance of Th17/Treg cells. Using in silico DNA copy number analysis, we have demonstrated that ~20% of CC samples exhibit gain of 8q22.3 and 19q13.31; the regions of the genome that encodes the KLF10 and PSG genes, respectively. Gene expression studies demonstrated that there were no alterations in KLF10 mRNA expression, whilst the PSG2 and -5 genes were up-regulated by 1.76 and 3.97-fold respectively in CC compared to normal tissue controls. siRNA and ChIP experiments in SiHa cells have demonstrated that KLF10 participates in immune response through regulation of IL6, IL25 and PSG2 and PSG5 genes. Using cervical tissues from KLF10-/- mice, we have identified down-regulation of PSG17, -21 and -23 and IL11. These results suggest that KLF10 may regulate immune system response genes in cervical cancer among other functions. KLF10 and PSG copy number variations and alterations in mRNA expression levels could represent novel molecular markers in CC.

Genotypic resistance mutations to antiretroviral drugs in HIV-1 B and non-B subtypes from Cuba / Mutaciones de resistencia genotípica a los fármacos antirretrovíricos en los subtipos B y no B del VIH-1 en Cuba

Objetivos. Determinar la prevalencia de la resistencia a los fármacos y analizar la presencia de mutaciones de resistencia genotípica en los subtipos B y no B del VIH-1 en Cuba. Métodos. Entre un total de 1 950 personas que se estima que están infectadas por el VIH-1 en Cuba, se estudió una muestra de 103 pacientes, de los cuales 76 habían recibido antirretrovíricos y 27 no. Se determinó la carga de ARN vírico en el plasma y se utilizó la secuenciación automatizada para detectar mutaciones de resistencia a los inhibidores de la transcriptasa inversa (ITI) y de la proteasa (IP). También se procedió a la subtipificación de la región V3 con prueba de movilidad de heteroduplex (HMA). Para confirmar los resultados de esta prueba se secuenció el gen env (C2-V3- C3) en un tercio de las muestras de cada uno de los subtipos detectados por HMA. Resultados. De las 103 muestras, 81 (78,6%) fueron clasificadas como pertenecientes al subtipo B, 19 (18,5%) al A y 3 (2,9%) al C. La prevalencia de mutaciones de resistencia fue del 26,2% para los ITI y 3,9% para los ITI más IP. Para los ITI nucleosídicos fue del 27,6% en los pacientes tratados y del 7,4% en los no tratados; para los ITI no nucleosídisos, las cifras correspondientes fueron del 5,3% y 0%, respectivamente. En los pacientes tratados se detectó una baja frecuencia (2,6%) de resistencia a la zidovudina más lamivudina y abacavir, y no se encontró multirresistencia a los ITI nucleosídicos. Para las combinaciones de IP e ITI, la prevalencia de la resistencia fue del 5,3% en los pacientes tratados y del 0% en los no tratados. Conclusiones. Aunque generalmente se considera que en Cuba predomina el subtipo B, en este estudio se detectó una alta proporción de virus del subtipo no B. La baja prevalencia de mutaciones de resistencia a los IP e ITI refleja la introducción más tardía de estos fármacos en el país. Como no se observó multirresistencia a los ITI, el empleo de estos fármacos sigue siendo una opción válida para los pacientes cubanos

Objetivos. Determinar la prevalencia de la resistencia a los fármacos y analizar la presencia de mutaciones de resistencia genotípica en los subtipos B y no B del VIH-1 en Cuba. Métodos. Entre un total de 1 950 personas que se estima que están infectadas por el VIH-1 en Cuba, se estudió una muestra de 103 pacientes, de los cuales 76 habían recibido antirretrovíricos y 27 no. Se determinó la carga de ARN vírico en el plasma y se utilizó la secuenciación automatizada para detectar mutaciones de resistencia a los inhibidores de la transcriptasa inversa (ITI) y de la proteasa (IP). También se procedió a la subtipificación de la región V3 con prueba de movilidad de heteroduplex (HMA). Para confirmar los resultados de esta prueba se secuenció el gen env (C2-V3- C3) en un tercio de las muestras de cada uno de los subtipos detectados por HMA. Resultados. De las 103 muestras, 81 (78,6%) fueron clasificadas como pertenecientes al subtipo B, 19 (18,5%) al A y 3 (2,9%) al C. La prevalencia de mutaciones de resistencia fue del 26,2% para los ITI y 3,9% para los ITI más IP. Para los ITI nucleosídicos fue del 27,6% en los pacientes tratados y del 7,4% en los no tratados; para los ITI no nucleosídisos, las cifras correspondientes fueron del 5,3% y 0%, respectivamente. En los pacientes tratados se detectó una baja frecuencia (2,6%) de resistencia a la zidovudina más lamivudina y abacavir, y no se encontró multirresistencia a los ITI nucleosídicos. Para las combinaciones de IP e ITI, la prevalencia de la resistencia fue del 5,3% en los pacientes tratados y del 0% en los no tratados. Conclusiones. Aunque generalmente se considera que en Cuba predomina el subtipo B, en este estudio se detectó una alta proporción de virus del subtipo no B. La baja prevalencia de mutaciones de resistencia a los IP e ITI refleja la introducción más tardía de estos fármacos en el país. Como no se observó multirresistencia a los ITI, el empleo de estos fármacos sigue siendo una opción válida para los pacientes cubanos