Early in infection, human immunodeficiency virus type 1 (HIV-1) generally uses the CCR5 chemokine receptor (along with CD4) for cellular entry. In many HIV-1-infected individuals, viral genotypic changes arise that allow the virus to use CXCR4 (either in addition to CCR5 or alone) as an entry coreceptor. This switch has been associated with an acceleration of both CD3؉ T-cell decline and progression to AIDS. While it is well known that the V3 loop of gp120 largely determines coreceptor usage and that positively charged residues in V3 play an important role, the process of genetic change in V3 leading to altered coreceptor usage is not well understood. Further, the methods for biological phenotyping of virus for research or clinical purposes are laborious, depend on sample availability, and present biosafety concerns, so reliable methods for sequencebased "virtual phenotyping" are desirable. We introduce a simple bioinformatic method of scoring V3 amino acid sequences that reliably predicts CXCR4 usage (sensitivity, 84%; specificity, 96%). This score (as determined on the basis of position-specific scoring matrices [PSSM]) can be interpreted as revealing a propensity to use CXCR4 as follows: known R5 viruses had low scores, R5X4 viruses had intermediate scores, and X4 viruses had high scores. Application of the PSSM scoring method to reconstructed virus phylogenies of 11 longitudinally sampled individuals revealed that the development of X4 viruses was generally gradual and involved the accumulation of multiple amino acid changes in V3. We found that X4 viruses were lost in two ways: by the dying off of an established X4 lineage or by mutation back to low-scoring V3 loops.Early studies of the biological properties of human immunodeficiency virus type 1 (HIV-1) found that virus isolates could be placed into as few as two phenotypic categories (defined in vitro as either non-syncytium-inducing [NSI] or syncytium-inducing [SI]) in certain CD4 ϩ T-cell lines. These phenotypes were often found to be associated with differences in growth properties and cytopathicity on peripheral blood mononuclear cells (PBMC) (1,14,46) and in cellular host range (3,48). Ultimately, the difference between the NSI and SI phenotypes was shown to be due largely to the differential use of chemokine receptors as coreceptors for viral entry: NSI viruses predominantly use CCR5, while SI viruses can use CCR5 and CXCR4 or CXCR4 exclusively (2, 29, 31, 52, 54). Results determined on the basis of SI phenotype and/or coreceptor usage typing showed that although HIV-1 present at primary infections used the CCR5 coreceptor (R5 virus) ϳ90% of the time (63, 67, 68), a substantial proportion of individuals eventually developed virus that used the CXCR4 coreceptor (X4 virus). These X4/SI viruses are associated with accelerated CD4 decline and more rapid progression of HIV-1 disease (8,28,33,43,47). Little is known about the mechanisms by which these viruses come to predominate among the HIV-1 strains present in an infected person. For example, it is no...

Epithelial sodium channels (ENaCs) perform diverse physiological roles by mediating Na+ absorption across epithelial surfaces throughout the body. Excessive Na+ absorption in kidney and colon elevates blood pressure and in the airways disrupts mucociliary clearance. Potential therapies for disorders of Na+ absorption require better understanding of ENaC regulation. Recent work has established partial and selective proteolysis of ENaCs as an important means of channel activation. In particular, channel-activating transmembrane serine proteases (CAPs) and cognate inhibitors may be important in tissue-specific regulation of ENaCs. Although CAP2 (TMPRSS4) requires catalytic activity to activate ENaCs, there is not yet evidence of ENaC fragments produced by this serine protease and/or identification of the site(s) where CAP2 cleaves ENaCs. Here, we report that CAP2 cleaves at multiple sites in all three ENaC subunits, including cleavage at a conserved basic residue located in the vicinity of the degenerin site (α-K561, β-R503, and γ-R515). Sites in α-ENaC at K149/R164/K169/R177 and furin-consensus sites in α-ENaC (R205/R231) and γ-ENaC (R138) are responsible for ENaC fragments observed in oocytes coexpressing CAP2. However, the only one of these demonstrated cleavage events that is relevant for the channel activation by CAP2 takes place in γ-ENaC at position R138, the previously identified furin-consensus cleavage site. Replacement of arginine by alanine or glutamine (α,β,γR138A/Q) completely abolished both the Na+ current (INa) and a 75-kD γ-ENaC fragment at the cell surface stimulated by CAP2. Replacement of γ-ENaC R138 with a conserved basic residue, lysine, preserved both the CAP2-induced INa and the 75-kD γ-ENaC fragment. These data strongly support a model where CAP2 activates ENaCs by cleaving at R138 in γ-ENaC.

Dyschromatosis universalis hereditaria (DUH) is an autosomal dominant pigmentary genodermatosis characterized by the presence of patches of hyperpigmentation and hypopigmented macules distributed over the body, with most cases reported in Asia. DUH is a heterogeneous disease and a small portion of patients carry the ABCB6 variant. In the present study, exome sequencing of four generations of a Chinese family with DUH identified a c.1761C>G (p.Ser587Arg) mutation in exon 15 of SAM and SH3 domain containing 1 (SASH1) that was found to co-segregate in some family members. Immunohistological analysis of biopsy specimens showed that SASH1 was diffusely distributed in all layers of the epidermis, suggesting increased transepithelial migration of melanocytes (MCs). The point mutation c.1761C>G of SASH1 was successfully induced in immortalized human melanocyte (PIG1) cells, which resulted in the downregulation of SASH1 expression. Bioinformatics analysis showed that mutated SASH1 downregulated thrombospondin 1 (THBS1) expression and inactivated transforming growth factor beta 1 (TGF-β1) signaling. TGF-β1 expression by PIG1cells was found to negatively regulate SASH1 protein expression. Transwell migration and wound-healing assays showed an increase in the migration and invasion capabilities of the cells carrying the mutation. Further, SASH1 mutations induced downregulation of melanin content. The study results suggest cross-talking between SASH1-TGF-β1 signaling, demonstrating the proposed MC migration modulation models and affecting melanin trafficking in the epithelium.

Cicadas form intimate symbioses with bacteria to obtain nutrients that are scarce in the xylem fluid they feed on. The obligate symbionts in cicadas are purportedly confined to specialized bacteriomes, but knowledge of bacterial communities associated with cicadas is limited. Bacterial communities in the bacteriomes and organs of reproductive, digestive and excretory systems of two cicada species (Platypleura kaempferi and Meimuna mongolica) were investigated using different methods, and the bacterial diversity and distribution patterns of dominant bacteria in different tissues were compared. Within each species, the bacterial communities of testes are significantly different from those of bacteriomes and ovaries. The dominant endosymbiont Candidatus Sulcia muelleri is found not only in the bacteriomes and reproductive organs, but also in the “filter chamber + conical segment” of both species. The transmission mode of this endosymbiont in the alimentary canal and its effect on physiological processes merits further study. A novel bacterium of Rhizobiales, showing ~80% similarity to Candidatus Hodgkinia cicadicola, is dominant in the bacteriomes and ovaries of P. kaempferi. Given that the genome of H. cicadicola exhibits rapid sequence evolution, it is possible that this novel bacterium is a related endosymbiont with beneficial trophic functions similar to that of H. cicadicola in some other cicadas. Failure to detect H. cicadicola in M. mongolica suggests that it has been subsequently replaced by another bacterium, a yeast or gut microbiota which compensates for the loss of H. cicadicola. The distribution of this novel Rhizobiales species in other cicadas and its identification require further investigation to help establish the definition of the bacterial genus Candidatus Hodgkinia and to provide more information on sequence divergence of related endosymbionts of cicadas. Our results highlight the complex bacterial communities of cicadas, and are informative for further studies of the interactions and co-evolution of insect-microbial symbioses in Cicadoidea.

Summary Many insects depend on symbiont(s) for survival. This is particularly the case for sap‐feeding hemipteran insects. In this study, we revealed that symbionts harboured in cicadas are diverse and complex, and the yeast‐like fungal symbionts (YLS) are present in most cicada species, but Hodgkinia is absent. During vertical transmission, Sulcia became swollen with the outer membrane drastically changed, while Hodgkinia became shrunken and changed from irregular to roughly spherical. Sulcia and/or Hodgkinia were exocytosed from the bacteriocytes to the intercellular space of bacteriomes, where they gathered together and were extruded to haemolymph. YLS and associated facultative symbiont(s) in the fat bodies were released to the haemolymph based on bacteriocyte disintegration. The obligate symbiont(s) were endocytosed and exocytosed successively by the epithelial plug cells of the terminal oocyte, while associated facultative symbiont(s), and possibly also YLS, may take a ‘free ride’ on the transmission of obligate symbiont(s) to gain entry into the oocyte. Then, the intermixed symbionts formed a characteristic ‘symbiont ball’ in the oocyte. Our results suggest that YLS in cicadas represent a new example of a relatively early stage of symbiogenesis in insects and contribute to a better understanding of the diversity and transmission mechanisms of symbionts in insects.

Although transovarial transmission of bacteriome-associated symbionts in hemipteran insects is extremely important for maintaining intimate host-symbiont associations, our knowledge of cellular mechanisms underlying the transmission process is quite limited. We investigated bacterial communities of salivary glands, bacteriomes, and digestive and reproductive organs and clarified the transovarial transmission of bacteriome-associated symbionts of the mountain-habitat specialist Pycna repanda using integrated methods. The bacterial communities among different gut tissues and those of bacteriomes of males and females both show similarity, whereas differences are exhibited among bacterial communities in testes and ovaries. The primary symbionts “Candidatus Sulcia muelleri” (hereafter “Ca. Sulcia”) and “Candidatus Hodgkinia cicadicola” (hereafter “Ca. Hodgkinia”) were not only restricted to but also dominant in the bacteriomes and ovaries. “Ca. Hodgkinia” cells in the bacteriomes of both sexes exhibited different colors by histological and electron microscopy. Also considering the results of a restriction fragment length polymorphism (RFLP)-based cloning approach, we hypothesize that “Ca. Hodgkinia” may have split into cytologically different cellular lineages within this cicada species. Regarding the dominant secondary symbionts, Rickettsia was detected in the salivary glands, digestive organs, and testes, whereas Arsenophonus was detected in the bacteriomes and ovaries. Our results show that Arsenophonus can coexist with “Ca. Sulcia” and “Ca. Hodgkinia” within bacteriomes and can be transovarially transmitted with these obligate symbionts together from mother to offspring in cicadas, but it is not harbored in the cytoplasm of “Ca. Sulcia.” The change in the shape of “Ca. Sulcia” and “Ca. Hodgkinia” during the transovarial transmission process is hypothesized to be related to the limited space and novel microenvironment. IMPORTANCE Cicadas establish an intimate symbiosis with microorganisms to obtain essential nutrients that are extremely deficient in host plant sap. Previous studies on bacterial communities of cicadas mainly focused on a few widely distributed species, but knowledge about mountain-habitat species is quite poor. We initially revealed the physical distribution of the primary symbionts “Ca. Sulcia” and “Ca. Hodgkinia” and the dominant secondary symbionts Rickettsia and Arsenophonus in the mountain-habitat specialist Pycna repanda and then clarified the transovarial transmission process of bacteriome-associated symbionts in this species. Our observations suggest that “Ca. Hodgkinia” may have split into cytologically distinct lineages within this cicada species, and related cicadas might have developed complex mechanisms for the vertical transmission of the bacteriome-associated symbionts. We also revealed that Arsenophonus can be transovarially transmitted in auchenorrhynchan insects when it is not harbored in the cytoplasm of other endosymbionts. Our results highlight transovarial transmission mechanisms of bacteriome-associated symbionts in sap-feeding insects.

Camponotus ants harbor the obligate intracellular endosymbiont Blochmannia in their midgut bacteriocytes, but little is known about intestinal bacteria living in the gut lumen. In this paper we reported the results of a survey of the intestinal microflora of Camponotus japonicus Mayr based on small-subunit rRNA genes (16S rRNAs) polymerase chain reaction (PCR)-restriction fragment-length polymorphism analysis of worker guts. From 107 clones, 11 different restriction fragment-length polymorphism profiles were identified, and sequences blasting analysis found these represent four types of bacteria. Most (91.6%) of the clones were "Candidatus Blochmannia", the obligate endosymbionts of Camponotus ants, and 6.5% of the clones were "Candidatus Serratia symbiotica", a secondary endosymbiont of aphids; the remaining 2% clones were Fructobacillus fructosus and uncultured Burkholderiales bacterium, respectively. These results show that the diversity of gut bacteria in C. japonicus was low. "Candidatus Serratia symbiotica" was identified from Camponotus ants for the first time, an interesting result because Blochmannia's closest bacterial relative is also in the genus Serratia. This discovery supports the scenario that consumption of aphid honeydew or tissue provides an initial step in the evolution of an advanced symbiosis, and suggests that Camponotus ant could acquire other secondary endosymbionts from Hemiptera host through their diet. In addition, Burkholderiales bacterium also was identified from the gut of C. japonicus for the first time, and whether it is a nitrogen-recycling endosymbiont in Camponotus ants needs to be investigated further.

Background: Ubiquitination of intracellular N termini lysines inhibits cleavage/stimulation of ␣,␤,␥ epithelial sodium channel (ENaC) extracellular domains. Results: The N terminus of ␥-ENaC attains secondary structure upon sensing membrane phospholipids. Basic residues promote ENaC cleavage. Conclusion: Ubiquitination obscures residues mediating allosteric stimulatory N-terminal structural transition that promotes ENaC cleavage/stimulation. Significance: This study presents a new mechanism of allosteric linkage between cytosolic signaling pathways and extracellular ENaC proteolysis.