Diversity and Localization of Bacterial Endosymbionts from Whitefly Species Collected in Brazil

Whiteflies (Hemiptera: Aleyrodidae) are sap-sucking insect pests, and some cause serious damage in agricultural crops by direct feeding and by transmitting plant viruses. Whiteflies maintain close associations with bacterial endosymbionts that can significantly influence their biology. All whitefly species harbor a primary endosymbiont, and a diverse array of secondary endosymbionts. In this study, we surveyed 34 whitefly populations collected from the states of Sao Paulo, Bahia, Minas Gerais and Parana in Brazil, for species identification and for infection with secondary endosymbionts. Sequencing the mitochondrial Cytochrome Oxidase I gene revealed the existence of five whitefly species: The sweetpotato whitefly Bemisia tabaci B biotype (recently termed Middle East-Asia Minor 1 or MEAM1), the greenhouse whitefly Trialeurodes vaporariorum, B. tabaci A biotype (recently termed New World 2 or NW2) collected only from Euphorbia, the Acacia whitefly Tetraleurodes acaciae and Bemisia tuberculata both were detected only on cassava. Sequencing rRNA genes showed that Hamiltonella and Rickettsia were highly prevalent in all MEAM1 populations, while Cardinium was close to fixation in only three populations. Surprisingly, some MEAM1 individuals and one NW2 population were infected with Fritschea. Arsenopnohus was the only endosymbiont detected in T. vaporariorum. In T. acaciae and B. tuberculata populations collected from cassava, Wolbachia was fixed in B. tuberculata and was highly prevalent in T. acaciae. Interestingly, while B. tuberculata was additionally infected with Arsenophonus, T. acaciae was infected with Cardinium and Fritschea. Fluorescence in situ hybridization analysis on representative individuals showed that Hamiltonella, Arsenopnohus and Fritschea were localized inside the bacteriome, Cardinium and Wolbachia exhibited dual localization patterns inside and outside the bacteriome, and Rickettsia showed strict localization outside the bacteriome. This study is the first survey of whitely populations collected in Brazil, and provides further insights into the complexity of infection with secondary endosymionts in whiteflies.     

The DAPK family: a structure–function analysis

DAP-kinase (DAPK) is the founding member of a family of highly related, death associated Ser/Thr kinases that belongs to the calmodulin (CaM)-regulated kinase superfamily. The family includes DRP-1 and ZIP-kinase (ZIPK), both of which share significant homology within the common N-terminal kinase domain, but differ in their extra-catalytic domains. Both DAPK and DRP-1 possess a conserved CaM autoregulatory domain, and are regulated by calcium-activated CaM and by an inhibitory auto-phosphorylation within the domain. ZIPK’s activity is independent of CaM but can be activated by DAPK. The three kinases share some common functions and substrates, such as induction of autophagy and phosphorylation of myosin regulatory light chain leading to membrane blebbing. Furthermore, all can function as tumor suppressors. However, they also each possess unique functions and intracellular localizations, which may arise from the divergence in structure in their respective C-termini. In this review we will introduce the DAPK family, and present a structure/function analysis for each individual member, and for the family as a whole. Emphasis will be placed on the various domains, and how they mediate interactions with additional proteins and/or regulation of kinase function.     

Endocytosis and Autophagy: Exploitation or Cooperation?

Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here.Autophagy is a lysosome-mediated pathway for the degradation of cytosolic proteins and organelles, which is essential for cell homeostasis, development, and for the prevention of several human diseases and infection (Choi et al. 2013). Importantly, autophagy cannot occur without an active lysosome. However, it is becoming increasingly recognized that the endosomal pathway plays a greater role than just providing the degradative enzymes found in the lysosome. Recent data suggest that in mammalian cells multiple contributions from several stages of the endocytic pathway are essential for efficient autophagy. Here we outline the autophagic pathway and then address the recent data on how different endosomal compartments contribute to autophagy, and the molecular machinery required for the interaction of the endosome and lysosome during the formation, and consumption of the autophagosome. Given the model emerging that the amino-acid-sensitive autophagic pathway originates from the endoplasmic reticulum (ER), several questions arise, including how do recognition and productive interaction occur between an ER-derived membrane and endosomes? How are these interactions mediated, and which are essential for efficient autophagy?     

A reference-quality,fully annotated genome from a Puerto Rican individual

Until 2019, the human genome was available in only one fully annotated version, GRCh38, which was the result of 18 years of continuous improvement and revision. Despite dramatic improvements in sequencing technology, no other genome was available as an annotated reference until 2019, when the genome of an Ashkenazi individual, Ash1, was released. In this study, we describe the assembly and annotation of a second individual genome, from a Puerto Rican individual whose DNA was collected as part of the Human Pangenome project. The new genome, called PR1, is the first true reference genome created from an individual of African descent. Due to recent improvements in both sequencing and assembly technology, and particularly to the use of the recently completed CHM13 human genome as a guide to assembly, PR1 is more complete and more contiguous than either GRCh38 or Ash1. Annotation revealed 37,755 genes (of which 19,999 are protein coding), including 12 additional gene copies that are present in PR1 and missing from CHM13. Fifty-seven genes have fewer copies in PR1 than in CHM13, 9 map only partially, and 3 genes (all noncoding) from CHM13 are entirely missing from PR1.     

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.     

Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon.

The characteristics of transmittance and fluorescence changes of 4-(p-aminostyryl)-1-pyridinium dyes in response to voltage-clamp pulses on the squid giant axon were examined. A zwitterionic styryl dye displays transmittance and excitation spectra on the voltage-clamped squid axon with shapes similar to those previously measured on a model membrane system and consistent with a postulated electrochromic mechanism. The speed of the transmittance response is faster than 1.2 microseconds. The size of the fluorescence change is a factor of 40 lower than on the model membrane; this diminution can be rationalized in terms of the background fluorescence from Schwann cells and the nonoptimal geometric arrangement of the axon membrane. When the emission spectrum is dissected from the excitation response, a nonelectrochromic component is found. This component might result from molecular motion during the excited state lifetime. A positively charged dye permeates the axon membrane and displays complex response waveforms dependent on the method of application and the axon holding potential. This contrasts markedly with model membrane results where the behavior of the cationic and zwitterionic dyes were indistinguishable.     

Polymorphism of Acetylcholinesterase in Discrete Regions of the Developing Human Fetal Brain

The molecular forms and membrane association of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) and pseudocholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) were determined in the presence of protease inhibitors in dissected regions of developing human fetal brain, as compared with parallel areas from mature brain. All areas contained substantial cholinesterase activities, of which acetylcholinesterase accounted for almost all the activity. Two major forms of acetylcholinesterase activity, sedimenting at 10-11S and 4-5S, respectively, were detected on sucrose gradients and possessed similar catalytic properties, as judged by their individual Km values toward [3H]acetylcholine (ca. 4 X 10(-4) M). The ratio between these forms varied by up to four- to fivefold, both between different areas and within particular areas at various developmental stages, but reached similar values (about 5:2) in all areas of mature brain. Acetylcholinesterase activity was ca. 35-50% low-salt-soluble and 45-65% detergent-soluble in various developmental stages and brain areas, with an increase during development of the detergent-soluble fraction of the light form. In contrast, pseudocholinesterase activity was mostly low-salt-soluble and sedimented as one component of 10-11S in all areas and developmental stages. Our findings suggest noncoordinate regulation of brain acetylcholinesterase and pseudocholinesterase, and indicate that the expression of acetylcholinesterase forms within embryonic brain areas depends both on cell type composition and on development.