Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis.

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

Functional morphology of the primary olfactory centers in the brain of the hermit crab Coenobita clypeatus (Anomala, Coenobitidae).

Terrestrial hermit crabs of the genus Coenobita display strong behavioral responses to volatile odors and are attracted by chemical cues of various potential food sources. Several aspects of their sense of aerial olfaction have been explored in recent years including behavioral aspects and structure of their peripheral and central olfactory pathway. Here, we use classical histological methods and immunohistochemistry against the neuropeptides orcokinin and allatostatin as well as synaptic proteins and serotonin to provide insights into the functional organization of their primary olfactory centers in the brain, the paired olfactory lobes. Our results show that orcokinin is present in the axons of olfactory sensory neurons, which target the olfactory lobe. Orcokinin is also present in a population of local olfactory interneurons, which may relay lateral inhibition across the array of olfactory glomeruli within the lobes. Extensive lateral connections of the glomeruli were also visualized using the histological silver impregnation method according to Holmes-Blest. This technique also revealed the structural organization of the output pathway of the olfactory system, the olfactory projection neurons, the axons of which target the lateral protocerebrum. Within the lobes, the course of their axons seems to be reorganized in an axon-sorting zone before they exit the system. Together with previous results, we combine our findings into a model on the functional organization of the olfactory system in these animals.

Mitochondrial genomes from Bronze Age Poland reveal genetic continuity from the Late Neolithic and additional genetic affinities with the steppe populations.

OBJECTIVE: In this work we aim to investigate the origins and genetic affinities of Bronze Age populations (2,400-1,100 BC) from the region of southern Poland and to trace maternal kinship patterns present in the burials of those populations by the use of complete mitochondrial genomes. MATERIALS AND METHODS: We performed ancient DNA analyses for Bronze Age individuals from present-day Poland associated with the Strzyzow culture, the Mierzanowice culture, and the Trzciniec Cultural circle. To obtain complete mitochondrial genomes, we sequenced genomic libraries using Illumina platform. Additionally, hybridization capture was used to enrich some of the samples for mitochondrial DNA. AMS 14 C-dating was conducted for 51 individuals to verify chronological and cultural attribution of the analyzed samples. RESULTS: Complete ancient mitochondrial genomes were generated for 80 of the Bronze Age individuals from present-day Poland. The results of the population genetic analyses indicate close maternal genetic affinity between Mierzanowice, Trzciniec, and Corded Ware culture-associated populations. This is in contrast to the genetically more distant Strzyzów people that displayed closer maternal genetic relation to steppe populations associated with the preceding Yamnaya culture and Catacomb culture, and with later Scythians. Potential maternal kinship relations were identified in burials of Mierzanowice and Trzciniec populations analyzed in this study. DISCUSSION: Results revealed genetic continuity from the Late Neolithic Corded Ware groups to Bronze Age Mierzanowice and Trzciniec-associated populations, and possible additional genetic contribution from the steppe to the formation of the Strzyzów-associated group at the end of 3rd millennium BC. Mitochondrial patterns indicated several pairs of potentially maternally related individuals mostly in Trzciniec-associated group.

Using expert-reviewed CSAM to train CNNs and its anthropological analysis.

Machine learning methods for the identification of child sexual abuse materials (CSAM) have been previously studied, however, they have serious limitations. Firstly, the training sets used to train the appropriate machine learning algorithms were not previously annotated by a forensic expert in anthropology. Secondly, previously presented solutions have rarely used models trained using real pornographic content involving children. Thirdly, previous studies have not presented a detailed justification for the classification decisions made, which is important due to the recent guidelines of the European Commission (Artificial Intelligence Act). The aim of the study was to train convolution neural networks (CNNs) using expert-labelled CSAM images and thereby identify the elements of the body and/or the environment that are critical for classifications by the neural network. To train and evaluate machine learning models, we used 60,000 images equally divided into four classes (CSAM images, images displaying sexual activity involving adults, images of people without sexual activity, and images not containing people). We used four neural network architectures: MobileNet, ResNet152, xResNet152 and its modification ResNet-s, designed for the purpose of research. The trained models provided high accuracy of classifying CSAM images: xResNet152 (F1 = 0.93, 92,8%), xResNet-s (F1 = 0.93, 93,1%), ResNet152 (F1 = 0.90, 91,39%), MobileNet (F1 ranged from 0.85 to 0.87, accuracy ranged from 86% to 87%). The results of the conducted research suggest that using expert knowledge (in sexology and anthropology) significantly improved the accuracy of the models. In regard to further anthropological analysis, the results indicate that the breasts, face and torso are crucial areas for the classification of pornographic content with children's participation. Results suggests that the ResNet-s neural network may be a reliable tool for clinical work and to support the work of experts witnesses in the field of anthropology. The study design received a positive opinion of the Ethics Committee of the Faculty of Mathematics, Informatics and Mechanics of the University of Warsaw. The clinical material was used for research purposes with the consent of the relevant prosecutor's offices. Authors provided free version of Windows application to classify CSAM for forensic experts, policemen and prosecutors at the OSF repository (DOI: 10.17605/OSF.IO/RU7JX).

Patrilocality and hunter-gatherer-related ancestry of populations in East-Central Europe during the Middle Bronze Age.

The demographic history of East-Central Europe after the Neolithic period remains poorly explored, despite this region being on the confluence of various ecological zones and cultural entities. Here, the descendants of societies associated with steppe pastoralists form Early Bronze Age were followed by Middle Bronze Age populations displaying unique characteristics. Particularly, the predominance of collective burials, the scale of which, was previously seen only in the Neolithic. The extent to which this re-emergence of older traditions is a result of genetic shift or social changes in the MBA is a subject of debate. Here by analysing 91 newly generated genomes from Bronze Age individuals from present Poland and Ukraine, we discovered that Middle Bronze Age populations were formed by an additional admixture event involving a population with relatively high proportions of genetic component associated with European hunter-gatherers and that their social structure was based on, primarily patrilocal, multigenerational kin-groups.

Molecular Oscillator Affects Susceptibility of Caterpillars to Insecticides: Studies on the Egyptian Cotton Leaf Worm-Spodoptera littoralis (Lepidoptera: Noctuidae).

The molecular oscillator is the core of the biological clock and is formed by genes and proteins whose cyclic expression is regulated in the transcriptional-translational feedback loops (TTFLs). Proteins of the TTFLs are regulators of both their own and executive genes involved in the control of many processes in insects (e.g., rhythmic metabolism of xenobiotics, including insecticides). We disrupted the clock operation in S. littoralis larvae by injecting the dsRNA of clock genes into their body cavity and culturing the larvae under continuous light. As a result, the daily susceptibility of larvae to insecticides was abolished and the susceptibility itself increased (in most cases). In the fat body, midgut, and Malpighian tubules (the main organs metabolizing xenobiotics) of the larvae treated with injected-dsRNA, the daily activity profiles of enzymes involved in detoxification-cytochrome P450 monooxygenases, Glutathione-S-transferase, and esterase-have changed significantly. The presented results prove the role of the molecular oscillator in the regulation of larvae responses to insecticides and provide grounds for rational use of these compounds (at suitable times of the day), and may indicate clock genes as potential targets of molecular manipulation to produce plant protection compounds based on the RNAi method.

Melatonin Lowers HIF-1α Content in Human Proximal Tubular Cells (HK-2) Due to Preventing Its Deacetylation by Sirtuin 1.

Although melatonin is widely known for its nephroprotective properties, there are no reports clearly pointing at its impact on the activity of hypoxia-inducible factor-1 (HIF-1), the main mediator of metabolic responses to hypoxia, in kidneys. The aim of the present study was to elucidate how melatonin affects the expression of the regulatory subunit HIF-1α in renal proximal tubules. HK-2 cells, immortalized human proximal tubular cells, were cultured under hypoxic conditions (1% O2). Melatonin was applied at 100 µM concentration. Protein and mRNA contents were determined by Western blot and RT-qPCR, respectively. HIF-1α acetylation level was established by means of immunoprecipitation followed by Western blot. Melatonin receptors MT1 and MT2 localization in HK-2 cells was visualized using immunofluorescence confocal analysis. It was found that melatonin in HK-2 cells (1) lowered HIF-1α protein, but not mRNA, content; (2) attenuated expression of HIF-1 target genes; (3) increased HIF-1α acetylation level; and (4) diminished sirtuin 1 expression (both protein and mRNA). Sirtuin 1 involvement in the regulation of HIF-1α level was confirmed applying cells with silenced Sirt1 gene. Moreover, the presence of membrane MT1 and MT2 receptors was identified in HK-2 cells and their ligand, ramelteon, turned out to mimic melatonin action on both HIF-1α and sirtuin 1 levels. Thus, it is concluded that the mechanism of melatonin-evoked decline in HIF-1α content in renal proximal tubular cells involves increased acetylation of this subunit which results from the attenuated expression of sirtuin 1, an enzyme reported to deacetylate HIF-1α. This observation provides a new insight to the understanding of melatonin action in kidneys.

[Replication of HCV in bone marrow of patients with haematological disorders]. / Replikacja wirusa zapalenia watroby typu C (HCV) w szpiku kostnym pacjentów z zaburzeniami hematologicznymi.

UNLABELLED: The aim of the study was to evaluate the presence of HCV replication in bone marrow cells derived from patients displaying hematological disorders. We analysed serum, peripheral blood mononuclear cells (PBMC) and bone marrow samples obtained from 27 patients displaying the following dysfunctions: lymphoma, trombocytopenia, haemophilia, pancytopenia and acute myeloid leukemia (AML). The presence of HCV-RNA in samples was detected by RT-PCR. All the serum samples were HCV-RNA positive as well as 9 out of 27 (33%) PBMC and 17 out of 27 (63%) of bone marrow samples. Independently to the disorder type, the co-presence of HCV-RNA in serum and bone marrow with the simultaneous absence of the viral genetic material in PBMC was detected in 5 (18.5%) of patients. This result suggests that bone marrow is a site of active viral replication. To check whether a viral replication generates any mutations, an SSCP analysis of the 5'UTR viral region was performed. The difference in the viral sequence derived from serum, PBMC and bone marrow was detected in one case. This result may indicate the occurrence of mutation process during the viral replication in bone marrow. An immunohistochemical analysis of bone marrow smears showed the presence of HCV antigens. CONCLUSION: bone marrow cells of patients displaying hematological disorders represent a putative site of extrahepatic HCV replication.

Temporal Expression of the Clock Genes in the Water Flea Daphnia pulex (Crustacea: Cladocera).

The timekeeping mechanisms that operate at the core of circadian clocks (oscillators) are based on interacting molecular feedback loops consisting of clock and clock-associated genes. However, there is a lack of comprehensive studies on the expression of clock genes (particularly those forming its core) in single crustacean species at the mRNA and protein levels, and these studies could serve as a basis for constructing a model of the crustacean molecular oscillator. Studies on Daphnia pulex are well suited to fill this gap because this species is the only representative crustacean whose genome has been sequenced. We analyzed the abundance of 20 gene transcripts throughout the day in the whole bodies of D. pulex (single clone); we found that 15 of these genes were transcriptionally active, and most had daily expression level changes. According to the functional classification of their homologues in insects, these genes may represent elements of the Daphnia molecular oscillator core and its input and output pathways. Studies of PERIOD (PER) protein, one of the main clock components, revealed its rhythmic expression pattern in the epidermis, gut, and ovaries. Finally, the cycling levels of many of these clock components observed in animals reared in continuous light led to the conclusion that the Daphnia oscillator, even if it is structurally similar to the oscillators of other arthropods, can be considered a particularly important adaptive mechanism for living in environments with extreme photoperiods.

Yolk proteins in the male reproductive system of the fruit fly Drosophila melanogaster: spatial and temporal patterns of expression.

In insects, spermatozoa develop in the testes as clones of single spermatogonia covered by specialized somatic cyst cells (cc). Upon completion of spermatogenesis, spermatozoa are released to the vas deferens, while the cc remain in the testes and die. In the fruit fly Drosophila melanogaster, the released spermatozoa first reach the seminal vesicles (SV), the organ where post-testicular maturation begins. Here, we demonstrate the temporal (restricted to the evening and early night hours) accumulation of membranous vesicles containing proteins in the SV lumen of D. melanogaster. When SV vesicles were isolated from the semen and co-incubated with testis-derived spermatozoa in vitro, their contents bound to the spermatozoa along their tails. The proteins of the SV vesicles were then characterized using 2-D electrophoresis. We identified a prominent protein spot of around 45-47 kDa, which disappears from the SV vesicles in the night, i.e. shortly after they appear in the SV lumen. Sequencing of peptides derived from this spot by mass spectrometry revealed identity with three yolk proteins (YP1-3). This unexpected result was confirmed by western blotting, which demonstrated that SV vesicles contain proteins that are immunoreactive with an antibody against D. melanogaster YP1-3. The expression of all yp genes was shown to be a unique feature of testis tissues. Using RNA probes we found that their transcripts localize exclusively to the cc that cover fully developed spermatozoa in the distal part of each testis. Temporally, the expression of yp genes was found to be restricted to a short period during the day and is followed by the evening accumulation of YP proteins in the cc. Immunohistochemical staining confirmed that cc are the source of SV vesicles containing YPs that are released into the SV lumen. These vesicles interact with spermatozoa and as a result, YPs become extrinsic proteins of the sperm membrane. Thus, we describe for the first time the expression of yolk proteins in the male reproductive system of D. melanogaster under physiological conditions, and show that somatic cells of the testes are the source of these proteins.

Neuropeptide complexity in the crustacean central olfactory pathway: immunolocalization of A-type allatostatins and RFamide-like peptides in the brain of a terrestrial hermit crab.

BACKGROUND: In the olfactory system of malacostracan crustaceans, axonal input from olfactory receptor neurons associated with aesthetascs on the animal's first pair of antennae target primary processing centers in the median brain, the olfactory lobes. The olfactory lobes are divided into cone-shaped synaptic areas, the olfactory glomeruli where afferents interact with local olfactory interneurons and olfactory projection neurons. The local olfactory interneurons display a large diversity of neurotransmitter phenotypes including biogenic amines and neuropeptides. Furthermore, the malacostracan olfactory glomeruli are regionalized into cap, subcap, and base regions and these compartments are defined by the projection patterns of the afferent olfactory receptor neurons, the local olfactory interneurons, and the olfactory projection neurons. We wanted to know how neurons expressing A-type allatostatins (A-ASTs; synonym dip-allatostatins) integrate into this system, a large family of neuropeptides that share the C-terminal motif -YXFGLamide. RESULTS: We used an antiserum that was raised against the A-type Diploptera punctata (Dip)-allatostatin I to analyse the distribution of this peptide in the brain of a terrestrial hermit crab, Coenobita clypeatus (Anomura, Coenobitidae). Allatostatin A-like immunoreactivity (ASTir) was widely distributed in the animal's brain, including the visual system, central complex and olfactory system. We focussed our analysis on the central olfactory pathway in which ASTir was abundant in the primary processing centers, the olfactory lobes, and also in the secondary centers, the hemiellipsoid bodies. In the olfactory lobes, we further explored the spatial relationship of olfactory interneurons with ASTir to interneurons that synthesize RFamide-like peptides. We found that these two peptides are present in distinct populations of local olfactory interneurons and that their synaptic fields within the olfactory glomeruli are also mostly distinct. CONCLUSIONS: We discuss our findings against the background of the known neurotransmitter complexity in the crustacean olfactory pathway and summarize what is now about the neuronal connectivity in the olfactory glomeruli. A-type allatostatins, in addition to their localization in protocerebral brain areas, seem to be involved in modulating the olfactory signal at the level of the deutocerebrum. They contribute to the complex local circuits within the crustacean olfactory glomeruli the connectivity within which as yet is completely unclear. Because the glomeruli of C. clypeatus display a distinct pattern of regionalization, their olfactory systems form an ideal model to explore the functional relevance of glomerular compartments and diversity of local olfactory interneurons for olfactory processing in crustaceans.

Diurnal rhythm in expression and release of yolk protein in the testis of Spodoptera littoralis (Lepidoptera: Noctuidae).

Circadian clocks (oscillators) regulate multiple life functions in insects. The circadian system located in the male reproductive tract of Lepidoptera is one of the best characterized peripheral oscillators in insects. Our previous research on the cotton leafworm, Spodoptera littoralis, demonstrated that this oscillator controls the rhythm of sperm release from the testis and coordinates sperm maturation in the upper vas deferens (UVD). We demonstrated previously that a protein that functions as yolk protein in females is also produced in cyst cells surrounding sperm bundles in the testis, and is released into the UVD. Here, we investigated the temporal expression of the yolk protein 2 (yp2) gene at the mRNA and protein level in the testis of S. littoralis, and inquired whether their expression is regulated by PER-based molecular oscillator. We describe a circadian rhythm of YP2 accumulation in the UVD seminal fluid, where this protein interacts with sperm in a circadian fashion. However, we also demonstrate that yp2 mRNA and YP2 protein levels within cyst cells show only a diurnal rhythm in light/dark (LD) cycles. These rhythms do not persist in constant darkness (DD), suggesting that they are non-circadian. Interestingly, the per gene mRNA and protein levels in cyst cells are rhythmic in LD but not in DD. Nevertheless, per appears to be involved in the diurnal timing of YP2 protein accumulation in cyst cells.

Clock-controlled rhythm of ecdysteroid levels in the haemolymph and testes, and its relation to sperm release in the Egyptian cotton leafworm, Spodoptera littoralis.

In Spodoptera littoralis, testicular sperm release occurs in a daily rhythm, which is controlled by endogenous circadian oscillator located in the male reproductive system. Although this rhythm is essential for male fertility, factors that initiate and maintain daily sperm release are not understood. In this study, we investigated a modulatory role for ecdysteroids in the sperm release rhythm and identified the source of ecdysteroids in adult males. We found that the onset of sperm release occurs two days pre-eclosion and coincides with a significant decrease in haemolymph ecdysteroids levels. 20-HE injection into the pupae prior to the first sperm release delayed its initiation and disrupted the developing rhythm in a dose dependent manner. 20-HE injection into adults depressed the number of sperm bundles leaving the testes. A day before the initial sperm release, ecdysteroid levels in the haemolymph and testes begin to oscillate in a circadian fashion. Ecdysteroid rhythms continue throughout imaginal life and correlate with the rhythm of sperm release. In each cycle, testicular sperm release coincides with a trough in testicular ecdysteroid concentration. Rhythmic changes in ecdysteroid levels are regulated by an endogenous circadian oscillator that continues to function in decapitated males. The generation of a complete cycle of ecdysteroid release by testes cultured in vitro indicates that this oscillator is located in the gonads. The haemolymph ecdysteroid levels are significantly lower and arrhythmic in males with removed testes, indicating that the testes are an important ecdysteroid source that may contribute to oscillations in haemolymph ecdysteroid levels.

Germ cell death in the testis and its relation to spermatogenesis in the wax moth, Galleria mellonella (Lepidoptera: Pyralidae), effects of facultative diapause.

The dichotomous spermatogenesis of many Lepidopterans results in the production of two types of sperm: eupyrene sperm possessing a cell nucleus which participates in fertilisation, and apyrene ones, which lose their nuclei during development and whose function remains a mystery. The goal of our study was to analyse spermatogenesis at the end of the larval development of the wax moth, Galleria mellonella, at an optimal temperature of 30 degrees C as well as to describe how they are affected by diapause brought on by a reduction of temperature to 18 degrees C. Spermatogenesis in non-diapausing insects did not differ significantly from that described in other species of Lepidoptera, and any differences found were compared against available literature. Based on the results presented, it may be unequivocally stated that changes in spermatogenesis occur in diapause caused by a suboptimal temperature of 18 degrees C. The main effect of diapause observed in the testes is the degeneration of germ cells, immediately following their differentiation from bipotential spermatocytes. Eupyrene cells seem to reach a more advanced stage of development. Due to the absence of secondary eupyrene spermatocytes in the testis of diapausing insects, it may be surmised that the meiotic divisions, which lead to the formation of secondary spermatocytes and eventually spermatids, do not occur, or are somehow altered. Lastly, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) analyses we performed show that the degenerative changes of eupyrene cells are apoptotic in character.

Relevance of lunar periodicity in human spontaneous abortions.

The effect of the moon on human reproduction has been scarcely investigated and with controversial results. The present analysis describes a significant effect of extreme perigeal lunar positions on the number of hospitalized spontaneous abortions (n = 1,329) recorded at two university clinics and a public hospital at Padova, Italy, during the years 2000-2003. Spectral analysis evidenced a 205-day period which appears to be correlated with the 206-day periodicity in extreme lunar distances. Circa-septan and circa-annual periodicities were also observed. Peak significances were determined by a Monte Carlo approach (circa-septan and 205-day periodicities: p < 0.001; circa-annual periodicity: p < 0.05). Our study indicates that the occurrence of human abortions displays suggestive periodicities that may be of relevance for gynecological and obstetrical practice.