Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases.

BACKGROUND: Anthrax, the zoonotic disease caused by the gram-positive bacterium Bacillus anthracis, is nowadays rare in northern parts of Europe including Finland and Scandinavia. Only two minor outbreaks of anthrax in 1988 and in 2004 and one sporadic infection in 2008 have been detected in animals in Finland since the 1970's. Here, we report on two Finnish B. anthracis strains that were isolated from spleen and liver of a diseased calf related to the outbreak in 1988 (strain HKI4363/88) and from a local scrotum and testicle infection of a bull in 2008 (strain BA2968). These infections occurred in two rural Finnish regions, i.e., Ostrobothnia in western Finland and Päijänne Tavastia in southern Finland, respectively. RESULTS: The isolates were genetically characterized by PCR-based methods such as multilocus variable number of tandem repeat analysis (MLVA) and whole genome-sequence analysis (WGS). Phylogenetic comparison of the two strains HKI4363/88 and BA2968 by chromosomal single nucleotide polymorphism (SNP) analysis grouped these organisms within their relatives of the minor canonical A-branch canSNP-group A.Br.003/004 (A.Br.V770) or canonical B-branch B.Br.001/002, respectively. Strain HKI4363/88 clustered relatively closely with other members of the A.Br.003/004 lineage from Europe, South Africa, and South America. In contrast, strain BA2968 clearly constituted a new sublineage within B.Br.001/002 with its closest relative being HYO01 from South Korea. CONCLUSIONS: Our results suggest that Finland harbors both unique (autochthonous) and more widely distributed, common clades of B. anthracis. We suspect that members of the common clades such as strains HKI4363/88 have been introduced only recently by anthropogenic activities involving importation of contaminated animal products. On the other hand, autochthonous strains such as isolate BA2968 probably have an older history of their introduction into Finland as evidenced by a high number of single nucleotide variant sites in their genomes.

The charged linker of the molecular chaperone Hsp90 modulates domain contacts and biological function.

The heat shock protein 90 (Hsp90) is a dimeric molecular chaperone essential in numerous cellular processes. Its three domains (N, M, and C) are connected via linkers that allow the rearrangement of domains during Hsp90's chaperone cycle. A unique linker, called charged linker (CL), connects the N- and M-domain of Hsp90. We used an integrated approach, combining single-molecule techniques and biochemical and in vivo methods, to study the unresolved structure and function of this region. Here we show that the CL facilitates intramolecular rearrangements on the milliseconds timescale between a state in which the N-domain is docked to the M-domain and a state in which the N-domain is more flexible. The docked conformation is stabilized by 1.1 kBT (2.7 kJ/mol) through binding of the CL to the N-domain of Hsp90. Docking and undocking of the CL affects the much slower intermolecular domain movement and Hsp90's chaperone cycle governing client activation, cell viability, and stress tolerance.

p23 and Aha1.

Hsp90 is a conserved molecular chaperone and is responsible for the folding and activation of several hundred client proteins, involved in various cellular processes. The large number and the diversity of these client proteins demand a high adaptiveness of Hsp90 towards the need of the individual client. This adaptiveness is amongst others mediated by more than 20 so-called cochaperones that differ in their actions towards Hsp90. Some of these cochaperones are able to modulate the ATPase activity of Hsp90 and/or its client protein binding, folding and activation. p23 and Aha1 are two prominent examples with opposing effects on the ATPase activity of Hsp90. p23 is able to inhibit the ATP turnover while Aha1 is the strongest known activator of the ATPase activity of Hsp90. Even though both cochaperones are conserved from yeast to man and have been studied for years, some Hsp90-related as well as Hsp90-independent functions are still enigmatic and under current investigation. In this chapter, we first introduce the ATPase cycle of Hsp90 and then focus on the two cochaperones integrating them in the Hsp90 cycle.

Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design.

Catching SARS-CoV-2 by Sequence Hybridization: a Comparative Analysis.

Controlling and monitoring the still ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic regarding geographical distribution, evolution, and emergence of new mutations of the SARS-CoV-2 virus is only possible due to continuous next-generation sequencing (NGS) and sharing sequence data worldwide. Efficient sequencing strategies enable the retrieval of increasing numbers of high-quality, full-length genomes and are, hence, indispensable. Two opposed enrichment methods, tiling multiplex PCR and sequence hybridization by bait capture, have been established for SARS-CoV-2 sequencing and are both frequently used, depending on the quality of the patient sample and the question at hand. Here, we focused on the evaluation of the sequence hybridization method by studying five commercially available sequence capture bait panels with regard to sensitivity and capture efficiency. We discovered the SARS-CoV-2-specific panel of Twist Bioscience to be the most efficient panel, followed by two respiratory panels from Twist Bioscience and Illumina, respectively. Our results provide on the one hand a decision basis for the sequencing community including a computation for using the full capacity of the flow cell and on the other hand potential improvements for the manufacturers. IMPORTANCE Sequencing the genomes of the circulating SARS-CoV-2 strains is the only way to monitor the viral spread and evolution of the virus. Two different approaches, namely, tiling multiplex PCR and sequence hybridization by bait capture, are commonly used to fulfill this task. This study describes for the first time a combined approach of droplet digital PCR (ddPCR) and NGS to evaluate five commercially available sequence capture panels targeting SARS-CoV-2. In doing so, we were able to determine the most sensitive and efficient capture panel, distinguish the mode of action of the various bait panels, and compute the number of read pairs needed to recover a high-quality full-length genome. By calculating the minimum number of read pairs needed, we are providing optimized flow cell loading conditions for all sequencing laboratories worldwide that are striving for maximizing sequencing output and simultaneously minimizing time, costs, and sequencing resources.

Structural elements in the flexible tail of the co-chaperone p23 coordinate client binding and progression of the Hsp90 chaperone cycle.

The co-chaperone p23 is a central part of the Hsp90 machinery. It stabilizes the closed conformation of Hsp90, inhibits its ATPase and is important for client maturation. Yet, how this is achieved has remained enigmatic. Here, we show that a tryptophan residue in the proximal region of the tail decelerates the ATPase by allosterically switching the conformation of the catalytic loop in Hsp90. We further show by NMR spectroscopy that the tail interacts with the Hsp90 client binding site via a conserved helix. This helical motif in the p23 tail also binds to the client protein glucocorticoid receptor (GR) in the free and Hsp90-bound form. In vivo experiments confirm the physiological importance of ATPase modulation and the role of the evolutionary conserved helical motif for GR activation in the cellular context.

Evaluation of a Highly Efficient DNA Extraction Method for Bacillus anthracis Endospores.

A variety of methods have been established in order to optimize the accessibility of DNA originating from Bacillus anthracis cells and endospores to facilitate highly sensitive molecular diagnostics. However, most endospore lysis techniques have not been evaluated in respect to their quantitative proficiencies. Here, we started by systematically assessing the efficiencies of 20 DNA extraction kits for vegetative B. anthracis cells. Of these, the Epicentre MasterPure kit gave the best DNA yields and quality suitable for further genomic analysis. Yet, none of the kits tested were able to extract reasonable quantities of DNA from cores of the endospores. Thus, we developed a mechanical endospore lysis protocol, facilitating the extraction of high-quality DNA. Transmission electron microscopy or the labelling of spores with the indicator dye propidium monoazide was utilized to assess lysis efficiency. Finally, the yield and quality of genomic spore DNA were quantified by PCR and they were found to be dependent on lysis matrix composition, instrumental parameters, and the method used for subsequent DNA purification. Our final standardized lysis and DNA extraction protocol allows for the quantitative detection of low levels (<50 CFU/mL) of B. anthracis endospores and it is suitable for direct quantification, even under resource-limited field conditions, where culturing is not an option.

Author Correction: A methylated lysine is a switch point for conformational communication in the chaperone Hsp90.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A methylated lysine is a switch point for conformational communication in the chaperone Hsp90.

Methylation of a conserved lysine in C-terminal domain of the molecular chaperone Hsp90 was shown previously to affect its in vivo function. However, the underlying mechanism remained elusive. Through a combined experimental and computational approach, this study shows that this site is very sensitive to sidechain modifications and crucial for Hsp90 activity in vitro and in vivo. Our results demonstrate that this particular lysine serves as a switch point for the regulation of Hsp90 functions by influencing its conformational cycle, ATPase activity, co-chaperone regulation, and client activation of yeast and human Hsp90. Incorporation of the methylated lysine via genetic code expansion specifically shows that upon modification, the conformational cycle of Hsp90 is altered. Molecular dynamics simulations including the methylated lysine suggest specific conformational changes that are propagated through Hsp90. Thus, methylation of the C-terminal lysine allows a precise allosteric tuning of Hsp90 activity via long distances.

First Phylogenetic Analysis of Malian SARS-CoV-2 Sequences Provides Molecular Insights into the Genomic Diversity of the Sahel Region.

We are currently facing a pandemic of COVID-19, caused by a spillover from an animal-originating coronavirus to humans occurring in the Wuhan region of China in December 2019. From China, the virus has spread to 188 countries and regions worldwide, reaching the Sahel region on March 2, 2020. Since whole genome sequencing (WGS) data is very crucial to understand the spreading dynamics of the ongoing pandemic, but only limited sequencing data is available from the Sahel region to date, we have focused our efforts on generating the first Malian sequencing data available. Screening 217 Malian patient samples for the presence of SARS-CoV-2 resulted in 38 positive isolates, from which 21 whole genome sequences were generated. Our analysis shows that both the early A (19B) and the later observed B (20A/C) clade are present in Mali, indicating multiple and independent introductions of SARS-CoV-2 to the Sahel region.

The identification of novel single nucleotide polymorphisms to assist in mapping the spread of Bacillus anthracis across the Southern Caucasus.

Anthrax is common as a zoonotic disease in the southern Caucasus area including parts of Turkey and Georgia. In this region, population genetics of the etiological agent Bacillus anthracis comprises, where known, the major canonical single nucleotide polymorphism (canSNP) groups A.Br.Aust94 and A.Br.008/009 of the pathogen's global phylogeny, respectively. Previously, isolates of B. anthracis from Turkey have been genotyped predominantly by multi locus variable number of tandem repeat analysis (MLVA) or canSNP typing. While whole genome sequencing is the future gold standard, it is currently still costly. For that reason we were interested in identifying novel SNPs which could assist in further distinguishing closely related isolates using low cost assay platforms. In this study we sequenced the genomes of seven B. anthracis strains collected from the Kars province of Eastern Anatolia in Turkey and discovered new SNPs which allowed us to assign these and other geographically related strains to three novel branches of the major A-branch canSNP-group (A.Br.) Aust94. These new branches were named Kafkas-Geo 1-3 and comprised isolates from the Kars region and the neighboring republic of Georgia suggesting a common ancestry. The novel SNPs identified in this study connect the population genetics of B. anthracis in the South Caucasus and Turkey and will likely assist efforts to map the spread of the pathogen across this region.

Allosteric Regulation Points Control the Conformational Dynamics of the Molecular Chaperone Hsp90.

Heat shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone responsible for the activation, maturation, and trafficking of several hundred client proteins in the cell. It is well known that (but not understood how) residues far away from Hsp90's nucleotide binding pocket can regulate its ATPase activity, a phenomenon called allosteric regulation. Here, the computational design of allosteric mutations was combined with in vitro and in vivo experiments to unravel nucleotide-responsive hot spots in the regulation of Hsp90. With this approach, we identified both activating and inhibiting regulation points and show that changes in those amino acids affect the conformational dynamics and ATPase activity of Hsp90 in vitro. Our observations that activating mutations loosen and inhibiting mutations rigidify the protein explain for the first time how Hsp90 changes in response to allosteric mutations. Additionally, mutations of these allosteric regulation points can be controlled by the interplay with Hsp90 co-chaperones, thus providing cells with an efficient mechanism of modifying Hsp90's intrinsic properties via different layers of regulation. Altogether, our results show that a framework for transmitting conformational information exists in the Hsp90 structure.

Chronic placental insufficiency affects retinal development in the guinea pig.

PURPOSE: Very low birth weight (VLBW) and fetal growth restriction are associated with increased risks of long-term visual impairments, including alterations to contrast sensitivity, a parameter mediated in part by dopaminergic amacrine cells. This study was conducted to determine whether chronic placental insufficiency (CPI), sufficient to cause growth restriction, results in neurochemical alterations to retinal interneurons, specifically amacrine and horizontal cell populations near term. METHODS: CPI was induced just before midgestation (term approximately 67 days of gestation, dg) in guinea pigs through unilateral ligation of the uterine artery. Growth-restricted (GR, n = 32) and control (n = 29) fetuses were euthanized at 60 dg and retinas prepared for analysis of amacrine cell populations by using antibodies to calbindin, calretinin, cholineacetyltransferase (ChAT), gamma-amino-butyric acid (GABA), dopamine beta-hydroxylase (D beta H), tyrosine hydroxylase (TH, dopaminergic), and NADPH-diaphorase histochemistry (nitrergic). Calbindin immunoreactivity (IR) was also used to identify horizontal cells. HPLC was used to assess concentrations of catecholamines and Western blot analysis to detect total TH levels. RESULTS: In GR compared with control fetuses the total number of TH-IR amacrine (P < 0.02) and calbindin-IR horizontal (P < 0.05) cells was reduced; however, there were no differences in the number of the ChAT, calbindin, calretinin, GABAergic, or nitrergic amacrine cell populations. HPLC revealed a reduction in the concentration of dopamine (P < 0.05) and noradrenaline (P < 0.05), and Western blot analysis revealed a reduction in TH in the retinas of GR compared with control fetuses (P < 0.05). CONCLUSIONS: CPI results in alterations to specific populations of retinal neurons. Such effects could contribute to visual impairments reported for VLBW children.

Altered retinal function and structure after chronic placental insufficiency.

PURPOSE: To consider whether growth restriction secondary to chronic placental insufficiency results in postnatal deficits in retinal structure and function. METHODS: Chronic placental insufficiency was induced just before midgestation in guinea pigs through unilateral ligation of the uterine artery. Eight weeks after birth, electroretinograms were recorded from prenatally compromised (PC, n = 6) and control (n = 15) animals. Data were collected for b-wave amplitude and implicit time, also the modeled receptoral (P3) response and oscillatory potentials were extracted. After electroretinography, retinas were prepared for structural analysis (PC, n = 6; control, n = 7). A separate cohort of PC (n = 8) and control (n = 9) animals underwent tyrosine hydroxylase immunoreactivity (TH-IR, dopaminergic neurons) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry (neuronal nitric oxide synthase, nNOS)--these being markers of amacrine cell subpopulations. RESULTS: Electroretinography revealed two PC guinea pigs with marked changes to saturated receptoral amplitude (Rm(P3)), sensitivity (log S) and postreceptoral waveforms. Grouped PC data revealed significantly reduced Rm(P3), whereas log S was not affected. The b-wave amplitudes were normal, but b-wave implicit times were delayed (P < 0.05) in PC animals. Amplitudes and peak times of oscillatory potentials were also significantly reduced and delayed (P < 0.05). Morphologic analysis revealed significant reductions in all cellular and plexiform (synaptic) layers in both the central (P < 0.05) and peripheral (P < 0.05) retina in PC animals. The outer retina, which contains the photoreceptors and the outer plexiform layer was particularly affected. The reduced growth of plexiform layers suggests a reduction in the growth of the neuropile in PC animals compared with control animals. The total number (P < 0.03) and density (P < 0.05) of TH-IR neurons was reduced, whereas the total number and density of nNOS-positive amacrine cells was not significantly different between PC and control animals. CONCLUSIONS: Chronic placental insufficiency results in morphologic and functional alterations to the retina. Electroretinogram deficits in PC animals indicated both inner and outer retinal anomalies. Such affects could contribute to the visual impairments reported in very-low-birth-weight children, some of whom are growth restricted.

Chronic placental insufficiency has long-term effects on auditory function in the guinea pig.

Very low birth weight and growth-restricted infants have an increased risk of auditory impairments. It is uncertain whether these impairments are related to adverse pre-, peri- or postnatal events. We aimed to determine whether a period of chronic placental insufficiency (CPI) in the guinea pig results in long-term alterations to auditory function. Near mid-gestation, CPI was induced via unilateral ligation of the uterine artery. At 8 weeks of age, auditory brainstem responses (ABRs) were recorded in response to unilateral acoustic stimulation in prenatally-compromised (PC, n=8) and control animals (n=8). Stimuli consisted of 100 micros clicks, presented at 33 pulses per second (pps) and tone pip stimuli at frequencies of 2, 4, 8, 16 and 32 kHz. To examine temporal response properties, click stimuli were also presented at rates of 66, 132 and 200 pps. Normal ABR waveforms were elicited by both click and tone pip stimuli in all animals. Moreover, there was no difference between control and PC animals in stimulus detection thresholds across the frequencies examined. Using high rate click stimuli, PC animals demonstrated a significant increase in both the latency of wave III (normalised to 33 pps) and the wave I-III inter-peak interval compared to the controls. We hypothesise that these functional changes reflect alterations in myelination of the auditory brainstem and/or changes in synaptic efficacy. The results suggest subtle deficits in neural conduction in the PC guinea pig at maturity, and may have implications for speech perception abilities of low birth weight or prenatally affected infants.

Intrauterine growth restriction affects the maturation of myelin.

Intrauterine growth-restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in the guinea pig results in long-term deficits in brain myelination and could therefore contribute to altered neural function. CPI was induced by unilateral ligation of the uterine artery at mid-gestation (term~67 days of gestation; dg), producing growth-restricted (GR) foetuses (60 dg), neonates (1 week) and young adults (8 week); controls were from the unligated horn or sham-operated animals. In GR foetuses (n=8) and neonates (n=7), white matter (WM) volume was reduced (p<0.05); this reduction did not persist in young adults (n=11) however the corpus callosum width was reduced (p<0.05). Immunoreactivity (IR) for myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and myelin proteolipid protein (PLP), all markers of myelinating oligodendrocytes (OL), was reduced in GR foetuses compared to controls. MBP was the most markedly affected with an abnormal retention of protein in the OL soma and a reduction of its incorporation into the myelin sheath. MAG-IR OL density was reduced (p<0.05), while the density of OLs immunoreactive for Olig-2, a transcription factor expressed throughout the entire OL lineage, was increased (p<0.05). MBP-, MAG- and PLP-IR recovered to control levels postnatally. These results suggest that IUGR transiently delays OL maturation and myelination in utero but that myelination and WM volume are restored to control levels postnatally. Long-term deficits in myelination are therefore unlikely to be the major factor underlying the altered neurological function which can be associated with IUGR.

Investigating the neurodevelopmental hypothesis of schizophrenia.

1. An optimal intra-uterine environment is critical for normal development of the brain. It is now thought that abnormal development in a compromised prenatal and/or early postnatal environment may be a risk factor for several neurological disorders that manifest postnatally, such as cerebral palsy, schizophrenia and epilepsy. 2. The present review examines some of the effects of abnormal prenatal brain development and focuses on one disorder that has been hypothesized to have, at least in part, an early neurodevelopmental aetiology: schizophrenia. 3. The key neuropathological alterations and changes in some of the neurotransmitter systems observed in patients with schizophrenia are reviewed. Evidence in support of a neurodevelopmental hypothesis for schizophrenia is examined. 4. A summary of the animal models that have been used by researchers in an attempt to elucidate the origins of this disorder is presented. Although no animal model of a complex human disorder is ever likely to emulate deficits in all aspects of structure and function observed in patients with a neuropsychiatric illness, our findings and those of others give support to the early neurodevelopmental hypothesis. 5. Thus, it is possible that an adverse event in utero disrupts normal brain development and creates a vulnerability of the brain that predisposes an already at-risk individual (e.g. genetic inheritance) to develop the disorder later in life.

Cardiovascular and renal disease in the adolescent guinea pig after chronic placental insufficiency.

OBJECTIVE: The aim of this study was to determine the long-term effects of chronic placental insufficiency on the metabolic state and organ structure in the fetal and adolescent guinea pig. STUDY DESIGN: The maternal uterine artery was ligated at day 28-30 to reduce placental function and restrict fetal growth. Whole body and tissue weights and plasma metabolites were determined at 60 days of gestation and 8 weeks of age; tissue structure was determined at the latter age in restricted and control offspring. RESULTS: Fetal growth restriction increased fibrosis in the heart and kidneys (P < .05), increased aortic wall thickening (P < .01), reduced the number of glomeruli in the kidneys (P < .05), and increased the plasma urea and chloride in adolescent offspring. CONCLUSION: This study demonstrates that diseases in the heart, aorta, and kidneys that result from an adverse prenatal environment are evident at adolescence and may contribute to subsequent adult disease.