Miracle-dz/UniFAR-FTUs huggingface dataset:
The three major types of CRISPR‐Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus

CRISPR‐Cas systems are small RNA‐based immune systems that protect prokaryotes from invaders such as viruses and plasmids. We have investigated the features and biogenesis of the CRISPR (cr)RNAs in Streptococcus thermophilus (Sth) strain DGCC7710, which possesses four different CRISPR‐Cas systems including representatives from the three major types of CRISPR‐Cas systems. Our results indicate that the crRNAs from each CRISPR locus are specifically processed into divergent crRNA species by Cas proteins (and non‐coding RNAs) associated with the respective locus. We find that the Csm Type III‐A and Cse Type I–E crRNAs are specifically processed by Cas6 and Cse3 (Cas6e), respectively, and retain an 8‐nucleotide CRISPR repeat sequence tag 5′ of the invader‐targeting sequence. The Cse Type I–E crRNAs also retain a 21‐nucleotide 3′ repeat tag. The crRNAs from the two Csn Type II‐A systems in Sth consist of a 5′‐truncated targeting sequence and a 3′ tag; however, these are distinct in size between the two. Moreover, the Csn1 (Cas9) protein associated with one Csn locus functions specifically in the production of crRNAs from that locus. Our findings indicate that multiple CRISPR‐Cas systems can function independently in crRNA biogenesis within a given organism – an important consideration in engineering coexisting CRISPR‐Cas pathways.
COMPUTED TOMOGRAPHIC IMAGING OF NASAL DISEASE IN 100 DOGS

One hundred dogs with nasal disease were evaluated by computed tomography (CT). Certain findings were significantly correlated with neoplasia. These included patchy areas of increased density within soft tissue opacity, destruction of part or all of the ethmoid bones, abnormal soft tissue in the retrobulbar spaces, destruction of one or both the lateral maxillae, destruction of the nasal bone or rostral dorsal maxilla, or hyperostosis of the lateral maxilla. Combinations of these findings were even more significant for neoplasia. No single finding or combination of findings was absolutely definitive of neoplasia. The results of this study are useful in indicating that biopsy results should be questioned if one or more of the above factors is present and a diagnosis of inflammation is made.
Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element

Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole‐genome analysis of a methicillin‐resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives of this strain. Of the multiple copies of IS256 found in the USA500 genome, one was inserted in the promoter sequence of repressor of toxins (Rot), a master transcriptional regulator responsible for the expression of virulence factors in S. aureus. We show that insertion into the rot promoter by IS256 results in the derepression of cytotoxin expression and increased virulence. Taken together, this work provides new insight into evolutionary strategies by which S. aureus is able to modify its virulence properties and demonstrates a novel mechanism by which horizontal gene transfer directly impacts virulence through altering toxin regulation.
The expression of antibiotic resistance genes in antibiotic‐producing bacteria

Antibiotic‐producing bacteria encode antibiotic resistance genes that protect them from the biologically active molecules that they produce. The expression of these genes needs to occur in a timely manner: either in advance of or concomitantly with biosynthesis. It appears that there have been at least two general solutions to this problem. In many cases, the expression of resistance genes is tightly linked to that of antibiotic biosynthetic genes. In others, the resistance genes can be induced by their cognate antibiotics or by intermediate molecules from their biosynthetic pathways. The regulatory mechanisms that couple resistance to antibiotic biosynthesis are mechanistically diverse and potentially relevant to the origins of clinical antibiotic resistance.
NudC Nudix hydrolase from Pseudomonas syringae, but not its counterpart from Pseudomonas aeruginosa, is a novel regulator of intracellular redox balance required for growth, motility and biofilm formation

Nudix pyrophosphatases, ubiquitous in all organisms, have not been well studied. Recent implications that some of them may be involved in response to stress and in pathogenesis indicate that they play important biological functions. We have investigated NudC Nudix proteins from the plant pathogen Pseudomonas syringae pv. tomato str. DC3000 and from the human pathogen Pseudomonas aeruginosa PAO1161. We found that these homologous enzymes are homodimeric and in vitro preferentially hydrolyse NADH. The P. syringae mutant strain deficient in NudC accumulated NADH and displayed significant defects in growth, motility and biofilm formation. The wild type copy of the nudC gene with its cognate promoter delivered in trans into the nudC mutant restored its fitness. However, introduction of the P. syringae nudC gene under the control of the strong tacp promoter into either P. syringae or P. aeruginosa cells had a toxic effect on both strains. Opposite to P. syringae NudC, the P. aeruginosa NudC deficiency as well as its overproduction had no visible impact on cells. Moreover, P. aeruginosa NudC does not compensate the lack of its counterpart in the P. syringae mutant. These results indicate that NudC from P. syringae, but not from P. aeruginosa is vital for bacteria.
The Brucella abortus virulence regulator, LovhK, is a sensor kinase in the general stress response signalling pathway

In the intracellular pathogen Brucella abortus, the general stress response (GSR) signalling system determines survival under acute stress conditions in vitro, and is required for long‐term residence in a mammalian host. To date, the identity of the Brucella sensor kinase(s) that function to perceive stress and directly activate GSR signalling have remained undefined. We demonstrate that the flavin‐binding sensor histidine kinase, LovhK (bab2_0652), functions as a primary B. abortus GSR sensor. LovhK rapidly and specifically phosphorylates the central GSR regulator, PhyR, and activates transcription of a set of genes that closely overlaps the known B. abortus GSR regulon. Deletion of lovhK severely compromises cell survival under defined oxidative and acid stress conditions. We further show that lovhK is required for cell survival during the early phase of mammalian cell infection and for establishment of long‐term residence in a mouse infection model. Finally, we present evidence that particular regions of primary structure within the two N‐terminal PAS domains of LovhK have distinct sensory roles under specific environmental conditions. This study elucidates new molecular components of a conserved signalling pathway that regulates B. abortus stress physiology and infection biology.
Dietary behaviour of an adolescent French male population

The dietary history method was used to determine the total energy and macronutrient intake, the distribution of daily energy from the different meals and the energy contribution of various food groups, in a population of 299 boys, aged between 10 and 15 years. The effects of age, the social status of the father (SPS), the ponderal index, the importance of breakfast or lunch, and the level of energy intake on dietary indicators, were studied. In our population, energy intake increased with increasing age but the energy contribution of macronutrients did not vary. The diet of boys whose fathers were in the upper SPS classes differed from the diet of those boys whose fathers were manual workers: for example they consumed less energy and pastries, but more animal protein (in percentage of energy intake), more dairy products, and more fruit. The ponderal index was negatively related to energy intake but positively related to protein intake, especially from meat and meat products. The energy intake from protein, animal protein and the energy contribution of dairy products, meat and meat products, fish and vegetables was lower among large eaters than among small eaters. The energy contribution of pastries, sugar and sweets was higher among large eaters. Similarily, the energy intake from morning and evening snacks was also higher among large eaters. It should be noted that boys who had a substantial breakfast took in less energy from all other meals (including snacks and main meals). Such informaton is necessary in order to develop an effective health programme.
Decreased Heart Rate in a Geriatric Client After Physical Therapy Intervention and Accommodation With the C-Leg

In this case report, a deconditioned, 82-year-old man underwent transfemoral amputation secondary to chronic infection after knee resurfacing. He was initially fit with a nonmicroprocessor knee as part of the preparatory prosthesis. Nine months after amputation, he was fit with a C-Leg as a component of the definitive prosthesis. As part of the first year of rehabilitation, the patient underwent four separate physical therapy (PT) programs that included strengthening, conditioning, and patient education interventions. Following accommodation on each of the two prostheses, the patient performed a distance-based, 75 m fastest possible walking speed test while wearing a heart rate (HR) monitor. HR, target HR, gait velocity, and physiologic cost index were measured or calculated to determine the physiologic outcomes of rehabilitation. There was a marked decrease in both resting and working HR after the fourth PT program and accommodation to the C-Leg. Target HR was also more appropriate after rehabilitation, which confirmed the decreased HR response. Because of the magnitude of HR reductions while resting and working, it is possible that the patient gave a submaximal effort in addition to experiencing a training effect. The physiologic cost index seemed to diminish the difference observed in HR because of the structure of the equation and was not found to be complementary to the HR data. As a result of PT intervention, the C-Leg knee prosthesis, and personal goals, the patient retained the ability to care for his dependent wife in the home.
SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.
Time does not heal all wounds: Identifying children suffering from psychological trauma

Traumatic exposure is a common experience among children and may have severe and long lasting effects on child’s mental well-being and development. This dissertation reports on children who have been exposed to one or more potentially traumatic events during their lives and focuses on identifying those children suffering from psychological trauma. The results presented in this thesis expand current knowledge with regard to the definition of a traumatic event and the different traumatic stress profiles between single trauma and child maltreatment. It is found that children may regularly develop severe posttraumatic stress symptoms in the absence of a traumatic event as defined by the DSM-IV-TR and that the effects of child maltreatment tend to extend PTSD. Furthermore, this dissertation yields easy-to-use screening tools that can be implemented by health providers in order to identify children suffering from psychological trauma. These screening tools may help clinicians in assigning appropriate follow-up diagnostic and clinical care. Although it is recognized that there are a myriad of challenges and issues in the field of childhood trauma that are still in need of thorough investigation, it is hoped that this dissertation will raise increased awareness of the psychological impact of childhood trauma and will lead to enhanced screening as routine part of contact. In the end, this will help to improve the identification of children suffering from psychological trauma.
Integration of the catalytic subunit activates deneddylase activity in vivo as final step in fungal COP9 signalosome assembly

The eight‐subunit COP9 signalosome (CSN) is conserved from filamentous fungi to humans and functions at the interface between cellular signalling and protein half‐life control. CSN consists of six PCI and two MPN domain proteins and forms a scaffold for additional interacting proteins. CSN controls protein stability in the ubiquitin‐proteasome system where the MPN domain CSN5/CsnE subunit inactivates cullin‐RING ligases. The CSN5/CsnE isopeptidase functions as deneddylase and removes the ubiquitin‐like protein Nedd8. The six PCI domain proteins of human CSN form a horseshoe‐like ring and all eight subunits are connected by a bundle of C‐terminal α‐helices. We show that single deletions of any csn subunit of Aspergillus nidulans resulted in the lack of deneddylase activity and identical defects in the coordination of development and secondary metabolism. The CSN1/CsnA N‐terminus is dispensable for deneddylase activity but required for asexual spore formation. Complex analyses in mutant strains revealed the presence of a seven‐subunit pre‐CSN without catalytic activity. Reconstitution experiments with crude extracts of deletion strains and recombinant proteins allowed the integration of CSN5/CsnE into pre‐CSN resulting in an active deneddylase. This supports a stable seven subunit pre‐CSN intermediate where deneddylase activation in vivo can be controlled by CSN5/CsnE integration as final assembly step.
The Plasmodium falciparum exportome contains non‐canonical PEXEL/HT proteins

The pathogenicity of Plasmodium falciparum is partly due to parasite‐induced host cell modifications. These modifications are facilitated by exported P. falciparum proteins, collectively referred to as the exportome. Export of several hundred proteins is mediated by the PEXEL/HT, a protease cleavage site. The PEXEL/HT is usually comprised of five amino acids, of which R at position 1, L at position 3 and E, D or Q at position 5 are conserved and important for export. Non‐canonical PEXEL/HTs with K or H at position 1 and/or I at position 3 are presently considered non‐functional. Here, we show that non‐canonical PEXEL/HT proteins are overrepresented in P. falciparum and other Plasmodium species. Furthermore, we show that non‐canonical PEXEL/HTs can be cleaved and can promote export in both a REX3 and a GBP reporter, but not in a KAHRP reporter, indicating that non‐canonical PEXEL/HTs are functional in concert with a supportive sequence environment. We then selected P. falciparum proteins with a non‐canonical PEXEL/HT and show that some of these proteins are exported and that their export depends on non‐canonical PEXEL/HTs. We conclude that PEXEL/HT plasticity is higher than appreciated and that non‐canonical PEXEL/HT proteins cannot categorically be excluded from Plasmodium exportome predictions.
A jack of all trades: the multiple roles of the unique essential second messenger cyclic di‐AMP

Second messengers are key components of many signal transduction pathways. In addition to cyclic AMP, ppGpp and cyclic di‐GMP, many bacteria use also cyclic di‐AMP as a second messenger. This molecule is synthesized by distinct classes of diadenylate cyclases and degraded by phosphodiesterases. The control of the intracellular c‐di‐AMP pool is very important since both a lack of this molecule and its accumulation can inhibit growth of the bacteria. In many firmicutes, c‐di‐AMP is essential, making it the only known essential second messenger. Cyclic di‐AMP is implicated in a variety of functions in the cell, including cell wall metabolism, potassium homeostasis, DNA repair and the control of gene expression. To understand the molecular mechanisms behind these functions, targets of c‐di‐AMP have been identified and characterized. Interestingly, c‐di‐AMP can bind both proteins and RNA molecules. Several proteins that interact with c‐di‐AMP are required to control the intracellular potassium concentration. In Bacillus subtilis, c‐di‐AMP also binds a riboswitch that controls the expression of a potassium transporter. Thus, c‐di‐AMP is the only known second messenger that controls a biological process by interacting with both a protein and the riboswitch that regulates its expression. Moreover, in Listeria monocytogenes c‐di‐AMP controls the activity of pyruvate carboxylase, an enzyme that is required to replenish the citric acid cycle. Here, we review the components of the c‐di‐AMP signaling system.
Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus

Excretion of cytoplasmic proteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG‐binding protein, which is secreted via the Sec‐pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan‐binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non‐excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.
Periplasmic superoxide dismutase SodCI of Salmonella binds peptidoglycan to remain tethered within the periplasm

Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell‐associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non‐covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI and allow the now tethered SodCII to function during infection.
Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion

Gonococci secrete chromosomal DNA into the extracellular environment using a type IV secretion system (T4SS). The secreted DNA acts in natural transformation and initiates biofilm development. Although the DNA and its effects are detectable, structural components of the T4SS are present at very low levels, suggestive of uncharacterized regulatory control. We sought to better characterize the expression and regulation of T4SS genes and found that the four operons containing T4SS genes are transcribed at very different levels. Increasing transcription of two of the operons through targeted promoter mutagenesis did not increase DNA secretion. The stability and steady‐state levels of two T4SS structural proteins were affected by a homolog of tail‐specific protease. An RNA switch was also identified that regulates translation of a third T4SS operon. The switch mechanism relies on two putative stem‐loop structures contained within the 5′ untranslated region of the transcript, one of which occludes the ribosome binding site and start codon. Mutational analysis of these stem loops supports a model in which induction of an alternative structure relieves repression. Taken together, these results identify multiple layers of regulation, including transcriptional, translational and post‐translational mechanisms controlling T4SS gene expression and DNA secretion.