Morphology and distribution of sensilla on antennae and mouthparts of the adult bruchid beetles, Bruchidius coreanus (Coleoptera: Bruchidae).

Bruchidius coreanus is a serious pest on Gleditsia sinensis Lam during seed storage, causing significant losses to their yield in southwest China. To gain insight into their behavioral mechanisms, the external morphology, ultrastructure, and distribution of sensilla on antennae, maxillary palps, and labial palps of both male and female B. coreanus were observed using a scanning electron microscope. The results revealed that both male and female adults had serrated antennae comprising a scape, a pedicel, and nine flagellomeres (F1-F9). There were eight types and seven subtypes of antenna sensilla observed in both sexes, including Böhm sensilla (BS), two subtypes of sensilla chaetica (SC1 and SC2), two subtypes of sensilla trichodea (ST1 and ST2), three subtypes of sensilla basiconica (SB1, SB2, and SB3), sensilla auricillica (SA), sensilla styloconicum (SS), capitate pegs (CP), and sensilla cavity (SCa). The average length of BS and ST (ST1 and ST2) showed significant differences between males and females. Furthermore, the number of SC (SC1 and SC2), ST1, and SCa differed significantly between the sexes. Four types of sensilla were found on the maxillary palps and labial palps, with the length of ST on these palps significantly differing between males and females. Additionally, SS on male labial palps was significantly longer than in females. The number of SC significantly differed between the male and female maxillary palps and labial palps, while ST and SS showed significant differences in the maxillary palps. These findings will contribute to further electrophysiological recording and behavioral research. RESEARCH HIGHLIGHTS: The external morphology and distribution of various sensilla on the antennae, maxillary palps, and labial palps of Bruchidius coreanus were described. Eight types and seven subtypes of antenna sensilla were observed on the antennae, while four types of sensilla were observed on the maxillary palps and labial palps. The capitate pegs were found exclusively on the antennae of female B. coreanus.

Descriptions of two new stick insect species of Cnipsomorpha Hennemann, Conle, Zhang & Liu (Phasmatodea) from China based on integrative taxonomy.

Accurate taxonomical identification is an extremely important basis for stick insect research, including evolutionary biology but also applied biology such as pest control. In addition, genetic methods are a valuable identification auxiliary technology at present. Therefore, this paper used morphological and molecular data to investigate five stick insect specimens from the genus Cnipsomorpha in Yunnan, successfully identifying two new species: Cnipsomorphayunnanensis Xu, Jiang & Yang, sp. nov. and C.yuxiensis Xu, Jiang & Yang, sp. nov. A phylogenetic tree was constructed through their 28S and COI genes in order to infer the phylogenetic position of the two new species. Photographs of the new species and a key to all known Cnipsomorpha species are provided.

Genotypic and phenotypic variability of 22q11.2 microdeletions - an institutional experience.

Patients with chromosome 22q11.2 deletion syndromes classically present with variable cardiac defects, parathyroid and thyroid gland hypoplasia, immunodeficiency and velopharyngeal insufficiency, developmental delay, intellectual disability, cognitive impairment, and psychiatric disorders. New technologies including chromosome microarray have identified smaller deletions in the 22q11.2 region. An increasing number of studies have reported patients presenting with various features harboring smaller 22q11.2 deletions, suggesting a need to better elucidate 22q11.2 deletions and their phenotypic contributions so that clinicians may better guide prognosis for families. We identified 16 pediatric patients at our institution harboring various 22q11.2 deletions detected by chromosomal microarray and report their clinical presentations. Findings include various neurodevelopmental delays with the most common one being attention deficit hyperactivity disorder (ADHD), one reported case of infant lethality, four cases of preterm birth, one case with dual diagnoses of 22q11.2 microdeletion and Down syndrome. We examined potential genotypic contributions of the deleted regions.

Ultrastructure of the Sensilla on the Antennae and Mouthparts of Bean Weevils, Megabruchidius dorsalis (Coleoptera: Bruchinae).

Megabruchidius dorsalis (Fåhraeus, 1839) (Coleoptera: Bruchinae) is an important pest that damages the seeds of Gleditsia L. (Fabaceae: Caesalpinioideae). This beetle searches for host plants with its sensory system. To further explore the mechanisms of host location and to understand the ultrastructure of M. dorsalis, we examined the morphology and distribution of its sensilla on the antennae and mouthparts of male and female adults, using scanning electron microscopy (SEM). Both male and female antennae are serrated and can be divided into scape, pedicel, and flagellum. There were seven types and eight subtypes of antennal sensilla, including Bohm bristles (BB), two subtypes of sensilla trichoid (ST1, ST2), two subtypes of sensilla chaetica (SC1, SC2), four subtypes of sensilla basiconic (SB1, SB2, SB3, SB4), sensilla cavity (SCa), sensilla auricillica (SA), and sensilla gemmiformium (SG). Five types of maxillary and labial palp sensilla in the mouthparts were observed: sensilla chaetica (SC), sensilla trichoidea (ST), sensilla styloconica (SSt), sensilla coeloconica (SCo), and sensilla digitiform (SD). No sexual dimorphism in sensilla type was observed, but there were variations between males and females in the numbers and distribution along the antennae. There were more SA in males than in females, while the number of ST sensilla in the maxillary palps were lower in males than in females. ST1 were most abundant in both sexes. We discussed potential function related to structure via comparisons with previous investigations of bruchids and other insects. Our results provide a theoretical basis for further studies on sensory physiological function, using semiochemicals as effective biological controls of M. dorsalis.

Phenotypes Associated with 16p11.2 Copy Number Gains and Losses at a Single Institution.

Chromosome 16p11.2 is one of the susceptible sites for recurrent copy number variations (CNVs) due to flanking near-identical segmental duplications. Five segmental duplications, named breakpoints 1 to 5 (BP1-BP5), have been defined as recombination hotspots within 16p11.2. Common CNVs on 16p11.2 include a proximal ~593 kb between BP4 and BP5, and a distal ~220 kb between BP2 and BP3. We performed a search for patients carrying 16p11.2 CNVs, as detected using chromosome microarray (CMA), in the Molecular Diagnostic Laboratory at the University of Texas Medical Branch (UTMB), in Galveston. From March 2013 through April 2018, a total of 1200 CMA results were generated for germline testing, and 14 patients tested positive for 16p11.2 CNVs, of whom 7 had proximal deletion, 2 had distal deletion, 4 had proximal duplication, and 1 had distal duplication. Herein, we provide detailed phenotype data for these patients. Our study results show that developmental delay, abnormal body weight, behavioral problems, and hypotonia are common phenotypes associated with 16p11.2 CNVs.

Discovery and optimization of 2-aminopyridine derivatives as novel and selective JAK2 inhibitors.

Janus kinases (JAKs) including JAK1, JAK2, JAK3, and TYK2 are members of a family of intracellular nonreceptor tyrosine kinases, which have been demonstrated to be critical in the cell signaling pathway and involved in inflammatory diseases and cancer. V617F mutation in JAK2 has been implicated in polycythaemia vera (PV), essential thrombocythaemia (ET) and myelofibrosis (MF). Here, we described the design, synthesis, and biological evaluation of a series of 2-aminopyridine derivatives. The results of enzymatic activity assays supported compound 16m-(R) as a potential and selective JAK2 inhibitor, which exhibited high inhibitory activity with an IC50 of 3 nM against JAK2, and 85- and 76-fold selectivity over JAK1 and JAK3, respectively. Structure-activity relationships (SAR) and mechanistic analysis demonstrated that 16m-(R) might be a promising selective JAK2 inhibitor for further study.

Genotypic and phenotypic variability of 22q11.2 microduplications: An institutional experience.

Duplications in the 22q11.2 region can cause 22q11.2 duplication syndrome and encompass a variety of phenotypes including developmental delays, facial abnormalities, cardiovascular defects, central nervous system delays, and other congenital abnormalities. However, the contribution of these contiguous duplicated regions to the clinical phenotypes has not been fully elucidated. In this study, we identified nine patients carrying different 22q11.2 microduplications detected by chromosomal microarray. Of these patients, seven pediatric patients presented with various clinical features including two neonate cases died shortly after birth, and two healthy adults. We examined region specific genotype-phenotype associations and found unpredictability associated with 22q11.2 duplications in these nine patients.

Design, synthesis and structure-activity relationship study of aminopyridine derivatives as novel inhibitors of Janus kinase 2.

Janus Kinase 2 (JAK2) is a kind of intracellular non-receptor protein tyrosine kinase and has been certified as an important target for the treatment of myeloproliferative neoplasms and rheumatoid arthritis. However, the low selectivity and potential safety issues restrict the clinical applications of JAK2 inhibitors. Here we found that crizotinib showed good inhibitory activity against JAK2 by enzymatic assays (IC50 = 27 nM). Then we carried out structure-based drug design and synthesized a series of compounds with an aminopyridine scaffold. Finally, compound 12k and 12l were identified as the promising inhibitors of JAK2, which exhibited high inhibitory activity (IC50 = 6 nM and 3 nM, respectively) and selectivity for JAK2 over JAK1 and JAK3, and showed potent antiproliferative activities toward HEL human erythroleukemia cells. Moreover, 12k suppressed symptoms of the collagen-induced arthritis (CIA) model in rats.

Lack of correlation between HERV-K expression and HIV-1 viral load in plasma specimens.

HERV-K viral RNA has been reported in plasma specimens of HIV-1 infected individuals. Emerging data support the regulation and functional interaction between HERV-K and HIV-1, which warrant development of accurate HERV-K assays to evaluate HERV-K activation. In this study, we examined HERV-K RNA expression after careful removal of "contaminating" cellular DNA using DNase I. We found that DNase I digestion effectively reduced HERV-K RT-PCR positive signal. We also found that levels of HERV-K expression did not correlate with HIV-1 viral load. Our study is in agreement with the published studies on HERV-K activation in HIV-1 positive plasma specimens, and in addition, calls for careful removal of cellular DNA to accurately evaluate HERV-K RNA expression.

Non-structural and nucleocapsid proteins of Punta Toro virus induce apoptosis of hepatocytes through both intrinsic and extrinsic pathways.

Punta Toro virus (PTV; family Bunyaviridae, genus Phlebovirus) causes severe hepatic damage through brisk apoptosis of hepatocytes. In the present study, two viral proteins encoded by the S segment of the viral genome, non-structural (NSs) and nucleocapsid protein (N), were examined for their roles in apoptosis. Expression of NSs in HepG2 cells led to apoptosis in 45% of transfected cells, and with N, 28%, on average. These levels represent a four- to an eightfold increase over cells transfected with the mutated protein vectors. Caspase-3, -8 and -9 activities were increased by N protein when compared with the control NC (P < 0.05), and by NSsA and NSsB, as compared to control NSsC (P < 0.01). Treatment of the transfected cells with caspase-8 or -9 inhibitors markedly decreased apoptosis. Neutralization of TNF-alpha or Fas ligand had no effect on apoptosis. These results indicate that both NSs and N are responsible for causing hepatocyte apoptosis by triggering the extrinsic caspase-8 and intrinsic caspase-9 pathways.

Localization of West Nile Virus in monkey brain: double staining antigens immunohistochemically of neurons, neuroglia cells and West Nile Virus.

West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.

Human Monoclonal Fab Antibodies Against West Nile Virus and its Neutralizing Activity Analyzed in Vitro and in Vivo.

The disease progression with West Nile virus (WNV) infection in humans leads to meningitis or encephalitis and may cause death, particularly among elderly and immunocompromised individuals. Passive immunity using immunoglobulins has shown efficacy in treating some patients with WNV infection, which makes the development of human anti-WNV antibodies significant. The goal of this study was to construct a Fab-specific phage display library against WNV, and to identify and select clones with neutralizing activities. Total RNA was extracted from peripheral blood lymphocytes (PBLs) of two immunized individuals, and RT-PCR was used to amplify the Fab fragments containing the heavy (V(H)) and light (V(L)) chains. The amplified genes were sequentially cloned into the recombinant antibody expression vector pComb3-H, and the Fab-specific phage display library was packaged with helper phage VCS-M13. Five rounds of panning were carried out with WNV E protein domain III, and then binding antibodies were selected by ELISA. Antigen binding specificity, complementarity determining region (CDR) sequence of V(H) and V(L), and neutralizing activity against WNV were analyzed in vitro and in vivo. Eight Fab monoclonal antibodies recognized E protein domain III from a library of 7×10(7) clones/ml. Of the eight, one (Fab 1), exhibited significant neutralizing activity, and completely blocked 100 pfu WNV infection in Vero cells at a concentration 160 µg/ml. In contrast, Fab 13 and Fab 25, showed weaker neutralizing activities, and modestly blocked 100 pfu WNV infections at concentrations of 320 µg/ml and 160 µg/ml, respectively. However, animal studies showed that Fab 1 failed to protect mice from death at the concentration of 160µg/ml indicating that the neutralizing potential of an antibody in vivo is determined by the strength of binding and the abundance of its epitope for the virion.

Characterization of cell-death pathways in Punta Toro virus-induced hepatocyte injury.

Punta Toro virus (PTV; genus Phlebovirus, family Bunyaviridae) causes apoptosis of hepatocytes in vivo in experimentally infected hamsters and in vitro in cultured HepG2 cells. Screening for expression of apoptosis-related genes has shown alterations in the genes for tumour necrosis factor-alpha (TNF-alpha) and the TNF receptor family. This study examined the roles of the TNF receptor-related extrinsic pathway and the Bcl-2 family-associated mitochondrial pathway in PTV-induced cell death. The effects of caspase inhibitors (caspIs) and TNF on cellular viability, virus replication, and morphological and biochemical changes in apoptosis were examined in HepG2 cells at different time points after infection with PTV (Adames strain). The results showed that caspIs dampened the virus-induced reduction in cellular viability, partially suppressed and delayed viral titres and antigen expression, and partially decreased the expression of apoptotic genes, caspase activities and DNA fragmentation. TNF treatment further decreased cellular viability after PTV infection and increased the level of apoptosis, whilst caspIs partially inhibited these effects. These findings indicate that TNF, caspase-8 and caspase-9 contribute to PTV-induced hepatocytic apoptosis and that additional mediators are probably also involved in this process. These mediators from different pathways correlated with one another and may be interlinked.

Phylogenetic relationships among sandfly fever group viruses (Phlebovirus: Bunyaviridae) based on the small genome segment.

The phleboviruses are more diverse in terms of arthropod vectors and antigenic relationships than most other genera of arthropod-borne viruses. In this study, 30 sandfly fever group viruses from the Naples, Sicilian, Punta Toro, Icoaraci and Frijoles serocomplexes were sequenced. Phylogenetic analyses were performed based on the sequence of the open reading frame for the nucleoprotein (N) and non-structural (NSs) protein genes of the small (S) segment. The five resultant genotypic lineages correlated with the serological grouping and were similar to analysis of M segment sequences. The sequence identity for N and NSs genes within the Sicilian, Naples, Punta Toro, Icoaraci and Frijoles serocomplexes was determined. The results indicated that genetic divergence for the S segment is lower than that for the M segment, suggesting that the S segment is more stable during evolution.

Antigenic and genetic relationships among Rift Valley fever virus and other selected members of the genus Phlebovirus (Bunyaviridae).

Preliminary serologic data indicated that two South American phleboviruses (Belterra virus [BELTV] and Icoaraci virus [ICOV]) may be related to Rift Valley fever virus (RVFV), an African phlebovirus that causes severe hepatitis and hemorrhagic fever in humans. To further define this relationship and to investigate the underlying genetic basis, comparative serologic and genetic sequence analyses were performed with RVFV and five other New World phleboviruses (ICOV, BELTV, Salobo virus, Joa virus, and Frijoles virus). Serologically, a one-way cross reaction was confirmed between antibodies against these New World viruses and RVFV antigen. In contrast, phylogenetic analysis demonstrated clear separation of these viruses from RVFV, into distinct phylogenies, based on sequences of the small, medium, and large RNA segments.

Morphological characterization of hantavirus HV114 by electron microscopy.

OBJECTIVE: This study was sought to investigate the propagation and morphogenesis of a new strain of hantavirus, HV114. METHODS: The urine of patient with epidemic hemorrhagic fever was inoculated to Vero E6 cells for the virus isolation. Electron microscopy was used to observe the isolated virus, HV114 and the variation of infected Vero E6 cells. RESULTS: According to our observations, the size (90-120 nm) of HV114 is smaller than that reported previously as 110- 160 nm. While ribosome-like particles associated with virions originating from rodent hantaviruses were not observed in HV114, virion budding was exhibited. It suggests that the dumbbell-shaped particles may generated from the process of virion budding. The budding processes suggest that there are several sites for HV114 assembly and maturation, including the host endoplasmic reticulum-Golgi compartment and the host plasma membranes. CONCLUSIONS: The HV114 isolated from the urine of the patient is differed from other hantaviruses which were isolated from rat organs. HV114 might undergo changes during the viral transmission process from rodents to humans.

West Nile virus infection of primary mouse neuronal and neuroglial cells: the role of astrocytes in chronic infection.

Primary cultures of embryonic murine neurons and newborn mouse astrocytes were inoculated with West Nile virus (WNV) strain NY385-99 to compare the pathogenesis of WNV infection in these types of CNS cells. Two different outcomes were observed. WNV infection in the neurons was rapidly progressive and destructive; within 5 days, all of the neurons were destroyed through apoptosis. WNV infection in the astrocytes evolved more slowly and did not seem to be highly lethal to the cells. The infected astrocytes continued to produce infectious virus (10(4.6)-10(6.5) PFU/mL) for 114 days, in a permissive, persistent infection. During this period, WNV antigen could be shown in the cytoplasm of the infected astrocytes by immunocytochemical assay, transmission electron microscopy of ultrathin sections, and in the cell culture medium by complement fixation test. Our results with this in vitro experimental murine cell model indicate that astrocytes can develop chronic or persistent infection with WNV, suggesting that these cells may play a role in the maintenance of WNV in the CNS.

Study of IgG subclass profiles of anti-HBs in populations with different HBV infection status.

To study IgG-specific subclasses of hepatitis B virus (HBV) surface antigen (anti-HBs), in different populations in Taiwan, a comparison was made between 104 chronic carriers (60 male and 44 female) and 439 recovered individuals (247 male and 192 female). Biochemical analyses of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were also performed. Among the 104 chronic carriers, 21 patients had abnormal ALT and AST levels (> 25 IU/ml). When comparing the patients with abnormal ALT and AST levels to chronic carriers with normal ALT and AST levels, no statistical difference was observed for anti-HBs levels (p > 0.05). The IgG subclass pattern of the relative anti-HBs IgG subclass titers was IgG1 > IgG3 = IgG4 in both chronic carriers and recovered individuals (p < 0.05). IgG1 is the predominant anti-HBs antibody after HBV infection, in either chronic carriers or in HBV-cured individuals. This finding is partly inconsistent with data reported from other group who suggested in individuals naturally infected, the anti-HBs IgG consists mainly of IgG3 and IgG1. In contrast to that of our previous studies of anti-HBe and anti-HBc, the mean OD values of anti-HBs total IgG, and all IgG subclasses except for IgG2, of either males or females, were significantly higher in recovered individuals than in chronic carriers, while the mean OD values of anti-HBe and anti-HBc were significantly higher in chronic carriers than in recovered individuals (P < 0.05). The IgG subclass profile of anti-HBs in chronic carriers was not changed with liver inflammation and was independent of sex and age, except in individuals with abnormal ALT and AST for whom anti-HBs IgG1 was not significantly higher than IgG3 (p > 0.05), in spite of that whose mean O.D. value is higher.

The study of IgG subclass profiles of anti-hbc in populations with different status of HBV infection.

To study IgG subclasses for the hepatitis B virus (HBV) core antigen (anti-HBc) in different populations, a comparison was made between 104 chronic carriers (60 male and 44 female) and 434 recovered individuals (247 male and 192 female). Biochemistry analyses of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were also performed. Among the 104 chronic carriers, 21 patients were found to be ALT and AST abnormal (> 25 IU/ml). After comparing these ALT and AST abnormal patients with other ALT and AST normal chronic carriers, no statistical difference was observed in the OD values of the anti-HBe (p > 0.05). The ELISA results showed the anti-HBc IgG subclass pattern was IgG1 > IgG3 > IgG4 in chronic carriers and IgG3 > IgG1 > IgG4 in recovered individuals (p < 0.05). This result suggests the IgG1/IgG3 ratio may be related with HBV status. However, in spite of the different anti-HBc IgG1/IgG3 patterns demonstrated in different populations, both anti-HBc IgG1 and IgG3 concentrations were significantly higher in chronic carriers (p < 0.05). Therefore, both the anti-HBc IgG1/IgG3 ratio and their amounts differed. They may play a significant role in chronic carriers and recovered individuals. The anti-HBc IgG subclass profiles of chronic carriers were not changed regardless of liver inflammation, and were independent of sex and age.

Apoptosis of hepatocytes caused by Punta Toro virus (Bunyaviridae: Phlebovirus) and its implication for Phlebovirus pathogenesis.

Experimental infection of hamsters with Punta Toro virus (PTV) produces a disease with clinical and pathological similarities to the severe human hemorrhagic fever caused by Rift Valley fever virus (RVFV), thus providing an animal model for RVFV pathogenesis. In this model, hepatocytic apoptosis is the main pathological component of liver injuries that are responsible for severe hemorrhagic manifestations. To further elucidate whether viral replication in hepatocytes directly causes apoptosis, we studied the morphological and biochemical changes of apoptosis in HepG2 cells at different time points after PTV infection. Cellular viability began to decrease 12 hours after infection compared with controls. Caspases 3/7 were activated significantly at 48 and 72 hours after infection, and phosphatidylserine translocation and DNA fragmentation were also detected at 48 and 72 hours. Cell cycle analysis by flow cytometry showed that infected HepG2 cells were arrested at G(0)/G(1) phase. Furthermore, virus titer increased with apoptosis progression, suggesting that viral replication is necessary for the apoptotic process. These results indicate that PTV infection alone, without a secondary inflammatory cellular reaction, induces hepatocytic apoptosis and suggest that future therapeutics for RVFV hemorrhagic disease might target inhibition of cellular apoptotic pathways during the acute infection.