Biochemical characterization and immunocytochemical localization of EM66, a novel peptide derived from secretogranin II, in the rat pituitary and adrenal glands.

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.     

Genome-Wide Analysis of Cold Adaptation in Indigenous Siberian Populations

Following the dispersal out of Africa, where hominins evolved in warm environments for millions of years, our species has colonised different climate zones of the world, including high latitudes and cold environments. The extent to which human habitation in (sub-)Arctic regions has been enabled by cultural buffering, short-term acclimatization and genetic adaptations is not clearly understood. Present day indigenous populations of Siberia show a number of phenotypic features, such as increased basal metabolic rate, low serum lipid levels and increased blood pressure that have been attributed to adaptation to the extreme cold climate. In this study we introduce a dataset of 200 individuals from ten indigenous Siberian populations that were genotyped for 730,525 SNPs across the genome to identify genes and non-coding regions that have undergone unusually rapid allele frequency and long-range haplotype homozygosity change in the recent past. At least three distinct population clusters could be identified among the Siberians, each of which showed a number of unique signals of selection. A region on chromosome 11 (chr11:66–69 Mb) contained the largest amount of clustering of significant signals and also the strongest signals in all the different selection tests performed. We present a list of candidate cold adaption genes that showed significant signals of positive selection with our strongest signals associated with genes involved in energy regulation and metabolism (CPT1A, LRP5, THADA) and vascular smooth muscle contraction (PRKG1). By employing a new method that paints phased chromosome chunks by their ancestry we distinguish local Siberian-specific long-range haplotype signals from those introduced by admixture.     

Writing In and Reading ICU Diaries: Qualitative Study of Families' Experience in the ICU

Purpose

Keeping an ICU patient diary has been reported to benefit the patient''s recovery. Here, we investigated the families'' experience with reading and writing in patient ICU diaries kept by both the family and the staff.

Methods

We conducted a qualitative study involving 32 semi-structured in-depth interviews of relatives of 26 patients (34% of all family members who visited patients) who met our ICU-diary criterion, i.e., ventilation for longer than 48 hours. Grounded theory was used to conceptualise the interview data via a three-step coding process (open coding, axial coding, and selective coding).

Results

Communicative, emotional, and humanising experiences emerged from our data. First, family members used the diaries to access, understand, and assimilate the medical information written in the diaries by staff members, and then to share this information with other family members. Second, the diaries enabled family members to maintain a connection with the patient by documenting their presence and expressing their love and affection. Additionally, families confided in the diaries to maintain hope. Finally, family members felt the diaries humanized the medical staff and patient.

Conclusions

Our findings indicate positive effects of diaries on family members. The diaries served as a powerful tool to deliver holistic patient- and family-centered care despite the potentially dehumanising ICU environment. The diaries made the family members aware of their valuable role in caring for the patient and enhanced their access to and comprehension of medical information. Diaries may play a major role in improving the well-being of ICU-patient families.     

Morphological and physiological responses of proliferating shoots of teak to temporary immersion and BA treatments

Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.     

The Genetic Origin of Daunians and the Pan-Mediterranean Southern Italian Iron Age Context

The geographical location and shape of Apulia, a narrow land stretching out in the sea at the South of Italy, made this region a Mediterranean crossroads connecting Western Europe and the Balkans. Such movements culminated at the beginning of the Iron Age with the Iapygian civilization which consisted of three cultures: Peucetians, Messapians, and Daunians. Among them, the Daunians left a peculiar cultural heritage, with one-of-a-kind stelae and pottery, but, despite the extensive archaeological literature, their origin has been lost to time. In order to shed light on this and to provide a genetic picture of Iron Age Southern Italy, we collected and sequenced human remains from three archaeological sites geographically located in Northern Apulia (the area historically inhabited by Daunians) and radiocarbon dated between 1157 and 275 calBCE. We find that Iron Age Apulian samples are still distant from the genetic variability of modern-day Apulians, they show a degree of genetic heterogeneity comparable with the cosmopolitan Republican and Imperial Roman civilization, even though a few kilometers and centuries separate them, and they are well inserted into the Iron Age Pan-Mediterranean genetic landscape. Our study provides for the first time a window on the genetic make-up of pre-Roman Apulia, whose increasing connectivity within the Mediterranean landscape, would have contributed to laying the foundation for modern genetic variability. In this light, the genetic profile of Daunians may be compatible with an at least partial autochthonous origin, with plausible contributions from the Balkan peninsula.     

Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide-P64k protein conjugates

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.     

The origins of global invasions of the German wasp (<Emphasis Type="Italic">Vespula germanica</Emphasis>) and its infection with four honey bee viruses

A successful control or eradication programme using biological control or genetically-mediated methods requires knowledge of the origin and the extent of wasp genetic diversity. Mitochondrial DNA variation in the native and invaded range of the social wasp Vespula germanica was used to examine intra-specific genetic variation and invasive source populations. We also examined wasps for the presence of four viruses found in honey bees: Acute bee paralysis virus, Deformed wing virus, Israeli acute paralysis virus and Kashmir bee virus. German wasps showed reduced genetic diversity in the invaded range compared to that of their native range. Populations in the introduced range are likely to have arrived from different source populations. All four viral honey bee pathogens were found in V. germanica, although they varied in their distribution and strain. Multiple introductions of German wasps have occurred for most invaded regions, though some populations are genetically homogenous. The differing locations of origin will guide researchers searching for biocontrol agents and the reduced genetic diversity may make these wasps a potentially viable target for control via gene drives.     

Beringian standstill and spread of Native American founders

Native Americans derive from a small number of Asian founders who likely arrived to the Americas via Beringia. However, additional details about the initial colonization of the Americas remain unclear. To investigate the pioneering phase in the Americas we analyzed a total of 623 complete mtDNAs from the Americas and Asia, including 20 new complete mtDNAs from the Americas and seven from Asia. This sequence data was used to direct high-resolution genotyping from 20 American and 26 Asian populations. Here we describe more genetic diversity within the founder population than was previously reported. The newly resolved phylogenetic structure suggests that ancestors of Native Americans paused when they reached Beringia, during which time New World founder lineages differentiated from their Asian sister-clades. This pause in movement was followed by a swift migration southward that distributed the founder types all the way to South America. The data also suggest more recent bi-directional gene flow between Siberia and the North American Arctic.