The role of the 5' sensing function of ribonuclease E in cyanobacteria.

RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5'-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5' sensing, contrasting to the alternative 'direct entry' mode, which is independent of monophosphorylated 5' ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5' sensing function of RNase E and mapped on a transcriptome-wide level 283 5'-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5' sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli.

[Management of polypharmacy in older people with multimorbidity]. / Management der Polypharmazie bei älteren Menschen mit Multimorbidität.

If the individual diagnoses of older people with multimorbidity are treated according to guidelines and by different specialists, confusing medication plans are sometimes the consequence. Therefore, a regular and structured drug evaluation is essential. As the life goals of patients can be very different, especially in older age, certain preliminary considerations should be made when starting, prescribing or discontinuing medication, taking into account the individual situation, including geriatric aspects. Updated so-called positive and negative lists provide assistance as to which medications are suitable or unsuitable for older people. Discontinuing certain medications when the life expectancy is reduced certainly makes sense but undertreatment of symptoms that cause distress to people, such as pain, should definitely be avoided.

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA.

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803.

A ubiquitous bone marrow reservoir of preexisting SARS-CoV-2-reactive memory CD4+ T lymphocytes in unexposed individuals.

Circulating, blood-borne SARS-CoV-2-reactive memory T cells in persons so far unexposed to SARS-CoV-2 or the vaccines have been described in 20-100% of the adult population. They are credited with determining the efficacy of the immune response in COVID-19. Here, we demonstrate the presence of preexisting memory CD4+ T cells reacting to peptides of the spike, membrane, or nucleocapsid proteins of SARS-CoV-2 in the bone marrow of all 17 persons investigated that had previously not been exposed to SARS-CoV-2 or one of the vaccines targeting it, with only 15 of these persons also having such cells detectable circulating in the blood. The preexisting SARS-CoV-2-reactive memory CD4+ T cells of the bone marrow are abundant and polyfunctional, with the phenotype of central memory T cells. They are tissue-resident, at least in those persons who do not have such cells in the blood, and about 30% of them express CD69. Bone marrow resident SARS-CoV-2-reactive memory CD4+ memory T cells are also abundant in vaccinated persons analyzed 10-168 days after 1°-4° vaccination. Apart from securing the bone marrow, preexisting cross-reactive memory CD4+ T cells may play an important role in shaping the systemic immune response to SARS-CoV-2 and the vaccines, and contribute essentially to the rapid establishment of long-lasting immunity provided by memory plasma cells, already upon primary infection.

Biallelic variants in ZNF142 lead to a syndromic neurodevelopmental disorder.

Biallelic variants of the gene encoding for the zinc-finger protein 142 (ZNF142) have recently been associated with intellectual disability (ID), speech impairment, seizures, and movement disorders in nine individuals from five families. In this study, we obtained phenotype and genotype information of 26 further individuals from 16 families. Among the 27 different ZNF142 variants identified in the total of 35 individuals only four were missense. Missense variants may give a milder phenotype by changing the local structure of ZF motifs as suggested by protein modeling; but this correlation should be validated in larger cohorts and pathogenicity of the missense variants should be investigated with functional studies. Clinical features of the 35 individuals suggest that biallelic ZNF142 variants lead to a syndromic neurodevelopmental disorder with mild to moderate ID, varying degrees of delay in language and gross motor development, early onset seizures, hypotonia, behavioral features, movement disorders, and facial dysmorphism. The differences in symptom frequencies observed in the unpublished individuals compared to those of published, and recognition of previously underemphasized facial features are likely to be due to the small sizes of the previous cohorts, which underlines the importance of larger cohorts for the phenotype descriptions of rare genetic disorders.

Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition.

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and the spontaneously occurred V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified mRNAs likely regulated jointly by RNase E and an sRNA and potential 3' end-derived sRNAs. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria.

Prevention of EAE by tolerogenic vaccination with PEGylated antigenic peptides.

BACKGROUND: Therapeutic treatment options for chronic autoimmune disorders such as multiple sclerosis (MS) rely largely on the use of non-specific immunosuppressive drugs, which are not able to cure the disease. Presently, approaches to induce antigen-specific tolerance as a therapeutic approach; for example, by peptide-based tolerogenic 'inverse' vaccines have regained great interest. We have previously shown that coupling of peptides to carriers can enhance their capacity to induce regulatory T cells in vivo. METHOD: In this present study, we investigated whether the tolerogenic potential of immunodominant myelin T-cell epitopes can be improved by conjugation to the synthetic carrier polyethylene glycol (PEG) in an experimental autoimmune encephalomyelitis (EAE) mouse model for chronic MS (MOG C57BL/6). RESULTS: Preventive administration of the PEGylated antigenic peptide could strongly suppress the development of EAE, accompanied by reduced immune cell infiltration in the central nervous system (CNS). Depletion of regulatory T cells (Tregs) abrogated the protective effect indicating that Tregs play a crucial role in induction of antigen-specific tolerance in EAE. Treatment during the acute phase of disease was safe and did not induce immune activation. However, treatment at the peak of disease did not affect the disease course, suggesting that either induction of Tregs does not occur in the highly inflamed situation, or that the immune system is refractory to regulation in this condition. CONCLUSION: PEGylation of antigenic peptides is an effective and feasible strategy to improve tolerogenic (Treg-inducing) peptide-based vaccines, but application for immunotherapy of overt disease might require modifications or combination therapies that simultaneously suppress effector mechanisms.

Homeostasis and Durability of T-Cell Memory-The Resting and the Restless T-Cell Memory.

The molecular basis of the persistence of experienced T lymphocytes, also known as "memory T lymphocytes," is still enigmatic. We are beginning to understand their considerable heterogeneity and topographic compartmentalization into memory T cells circulating through the body and those residing in a particular tissue. In some tissues, like murine spleen, subpopulations of memory T cells proliferating in the absence of antigen (homeostatic proliferation) have been described. Other populations are maintained resting in terms of transcription, mobility, and proliferation in dedicated survival niches organized by stromal cells. The survival of these memory T cells is conditional on being in such a niche, where they can persist for a lifetime. Circulating memory T lymphocytes of distinct immune responses slowly decline in numbers over time. The rules governing their entry into and exit from blood, as well as their lifestyle outside of the blood and their relation to resident memory T cells are poorly understood. Homeostasis of circulating, proliferating, and resting memory T cells is obviously controlled by different rheostats: tissue-exit and tissue-entry signals for circulating and proliferation-inducing signals for proliferating memory T cells. For tissue-resident, resting memory T cells, it is the availability of their survival niche. Apparently, this mechanism (i.e., the link between memory T cell and stromal cell) is so robust that it provides efficient T-cell memory over a lifetime in tissues such as the bone marrow.

[Fluid management in older persons]. / Volumenmanagement im Alter.

Both exsiccosis and hydropic decompensation occur more frequently in the older persons and are associated with complications, increased morbidity and mortality. This paper presents the age-associated causes and consequences of both conditions, as well as a practical approach to preventive measures, diagnostics, and therapy.

[Treatment of geriatric patients with end-stage renal failure : Dialysis or conservative procedure?] / Versorgung des geriatrischen Patienten mit terminaler Niereninsuffizienz : Dialyse oder eher konservatives Prozedere?

Compared to younger patients, an acceptable state of health in older patients with various comorbidities is rarely achieved by the initiation of dialysis. Despite dialysis treatment, further functional and cognitive impairments often rapidly occur in geriatric patients. Thus, newer studies are concerned with the quality of life of this patient group after initiation of dialysis as well as with palliative treatment strategies as alternatives. A structured clarification for the patients on all possibilities with mediation of all necessary information is a prerequisite for a shared decision-making. To assess life expectancy after dialysis initiation, various scores have been developed but the sensitivity could not fulfil the expectations. In the case of renal replacement, chronic intermittent hemodialysis is the treatment form most frequently performed in geriatric patients. The main concern of conservative palliative treatment is the quality of life and the management of uremic symptoms, which have to be addressed by a multidisciplinary team.

Tolerogenic Immunomodulation by PEGylated Antigenic Peptides.

Current treatments for autoimmune disorders rely on non-specific immunomodulatory and global immunosuppressive drugs, which show a variable degree of efficiency and are often accompanied by side effects. In contrast, strategies aiming at inducing antigen-specific tolerance promise an exclusive specificity of the immunomodulation. However, although successful in experimental models, peptide-based tolerogenic "inverse" vaccines have largely failed to show efficacy in clinical trials. Recent studies showed that repetitive T cell epitopes, coupling of peptides to autologous cells, or peptides coupled to nanoparticles can improve the tolerogenic efficacy of peptides, suggesting that size and biophysical properties of antigen constructs affect the induction of tolerance. As these materials bear hurdles with respect to preparation or regulatory aspects, we wondered whether conjugation of peptides to the well-established and clinically proven synthetic material polyethylene glycol (PEG) might also work. We here coupled the T cell epitope OVA323-339 to polyethylene glycols of different size and structure and tested the impact of these nano-sized constructs on regulatory (Treg) and effector T cells in the DO11.10 adoptive transfer mouse model. Systemic vaccination with PEGylated peptides resulted in highly increased frequencies of Foxp3+ Tregs and reduced frequencies of antigen-specific T cells producing pro-inflammatory TNF compared to vaccination with the native peptide. PEGylation was found to extend the bioavailability of the model peptide. Both tolerogenicity and bioavailability were dependent on PEG size and structure. In conclusion, PEGylation of antigenic peptides is an effective and feasible strategy to improve Treg-inducing, peptide-based vaccines with potential use for the treatment of autoimmune diseases, allergies, and transplant rejection.

Impact of iron oxide nanoparticles on xenobiotic metabolism in HepaRG cells.

Iron oxide nanoparticles are used in various industrial fields, as a tool in biomedicine as well as in food colorants, and can therefore reach human metabolism via oral uptake or injection. However, their effects on the human body, especially the liver as one of the first target organs is still under elucidation. Here, we studied the influence of different representative iron oxide materials on xenobiotic metabolism of HepaRG cells. These included four iron oxide nanoparticles, one commercially available yellow food pigment (E172), and non-particulate ionic control FeSO4. The nanoparticles had different chemical and crystalline structures and differed in size and shape and were used at a concentration of 50 µg Fe/mL. We found that various CYP enzymes were downregulated by some but not all iron oxide nanoparticles, with the Fe3O4-particle, both γ-Fe2O3-particles, and FeSO4 exhibiting the strongest effects, the yellow food pigment E172 showing a minor effect and an α-Fe2O3 nanoparticle leading to almost no inhibition of phase I machinery. The downregulation was seen at the mRNA, protein expression, and activity levels. Thereby, no dependency on the size or chemical structure was found. This underlines the difficulty of the grouping of nanomaterials regarding their physiological impact, suggesting that every iron oxide nanoparticle species needs to be evaluated in a case-by-case approach.

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1-4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle7.

Specific microbiota enhances intestinal IgA levels by inducing TGF-ß in T follicular helper cells of Peyer's patches in mice.

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-ß, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the generation and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-ß expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer's patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-ß and of mucosal IgA.

Evolutionary Response to Climate Change in Migratory Pied Flycatchers.

Climate change is rapidly advancing spring phenology [1-3] but at different rates in different species [1, 4]. Whether these advances are solely driven by phenotypic plasticity [2, 5] or also involve evolution is hotly debated (e.g., [5-7]). In some species, including avian long-distance migrants, plastic responses to early springs may be constrained by inherited circannual timing programs [8, 9], making evolutionary adjustment the only viable mechanism for keeping pace with shifting phenology [5, 10]. This constraint may be contributing to population declines in migratory species [5, 10-12]. To test whether a migrant's timing program has evolved [10, 12], we replicated an experimental study of the annual cycle of long-distance migratory pied flycatchers (Ficedula hypoleuca) after 21 years of warming. Flycatchers are a model for studying constrained ecological responses to climate change [6, 10, 12, 13]. We show that the phase of the flycatcher circannual clock controlling spring moult, migration, and reproductive timing advanced by 9 days. A nearby wild population mirrored these changes, concurrently advancing egg-laying by 11 days. Furthermore, the time window during which wild flycatcher reproductive timing was most sensitive to ambient temperature advanced by 0.8 days year-1. These results support a role of phenotypic evolution [14] in changing spring phenology [15, 16]. We suggest that the timing programs of long-distance migratory birds may have greater adaptive potential than previously thought, leaving some scope for evolutionary rescue in a changing climate.

The relevant psychological burden of having a benign brain tumor: a prospective study of patients undergoing surgical treatment of cranial meningiomas.

OBJECTIVE: Meningiomas are the most common intracranial neoplasm. Evidence concerning surgical management and outcome is abundant, while the implications for the quality of life (QOL) of a patient confronted with the diagnosis and undergoing surgery are unclear. The authors conducted a prospective study to evaluate QOL in relation to psychological comorbidities comorbidities. METHODS: A prospective study of patients undergoing elective surgery for the removal of an intracranial meningioma was performed. The authors evaluated depression (Allgemeine Depressionsskala K score) and anxiety (Post-Traumatic Stress Scale-10 [PTSS-10]; State Trait Anxiety Inventory-State Anxiety and -Trait Anxiety [STAI-S and STAI-T]; and Anxiety Sensitivity Index-3 [ASI-3]) scores before surgery and at 3 and 12 months after surgery. The correlation between preoperative psychological burden and postoperative QOL as measured by the 36-Item Short Form Health Survey and EQ-5L questionnaires was analyzed. Incidence and influence of these psychiatric comorbidities on clinical outcome were examined. RESULTS: A total of 78 patients undergoing resection of a meningioma between January 2013 and September 2017 participated in the preoperative psychological screening and 71 patients fully completed postoperative follow-up examination after 1 year of follow-up. At presentation, 48 patients (67.7%) had abnormal anxiety scores, which decreased to 29.6% (p = 0.003). On follow-up at 12 months, mean EQ-5L visual analog scale scores were significantly lower in patients with pathological scores on the PTSS-10 (0.84 vs 0.69; p = 0.004), STAI-S (0.86 vs 0.68; p = 0.001), and STAI-T (0.85 vs 0.71; p = 0.011). Neurological status (modified Rankin Scale) improved slightly and showed some correlation with psychological comorbidities QOL scores (p = 0.167). There was a nonsignificant increase of EQ-5L scores over the period of follow-up (p = 0.174) in the repeated-measures analysis. In the regression analysis, impaired QOL and physical disability on follow-up correlated with elevated preoperative anxiety and depression levels. CONCLUSIONS: The QOL and physical disability of patients undergoing resection of an intracranial meningioma highly depend on preoperative anxiety and depression levels. Stress and anxiety scores generally decrease after the resection, which leads us to conclude that there is a tremendous emotional burden caused by an upcoming surgery, necessitating close psychooncological support in order to uphold functional outcome and health-related QOL in the postoperative course.

The role of wrist monitors to measure blood pressure in older adults.

BACKGROUND: Arterial hypertension is a common condition in older adults with increasing data about blood pressure (BP) targets and antihypertensive treatment in this population. Recent studies have opened new discussions about the different usual and unusual methods of blood pressure monitoring. However, there are no evidence-based recommendations whether BP should be measured at upper arms or at wrist, which seems to be more comfortable in older and frail people. AIMS: The purpose of this study was to test the quality of wrist BP monitors as diagnostic method in older adults. METHODS: BP measurements at both upper arms and at both wrists were compared under standardized conditions in 605 patients ≥ 75 years. Differences in wrist and upper arm BP were, furthermore, correlated with various diagnoses and parameters including ankle-brachial-index (ABI). RESULTS: In patients of 75-80 years, there were no differences in BP measurements at upper arms compared to wrists whereas in patients > 80 years, BP measurements at wrists were significantly lower than at upper arms. In both age groups BP measured at wrist was significantly lower in patients with ABI < 0.9. CONCLUSIONS: BP wrist monitors could be considered as a serious alternative in adults of 75-80 years or in older persons with normal ABI values.

[2018 ESC/ESH Guidelines for the Management of Arterial Hypertension: What's New with Regard to Our Older People?] / Neue europäische Empfehlungen zum Blutdruckmanagement im Alter.

In the "2018 ESC/ESH Guidelines for the management of arterial hypertension" there are new blood pressure (BP) recommendations with regard to older patients based on randomized controlled trials and well-conducted meta-analyses performed during the last years.It is recommended to base the diagnosis of hypertension on repeated office BP measurements under standardized conditions and in older people in out-of-office BP measurement with ABPM and/or HBPM if logistically and economically feasible. Furthermore lying and standing BP measurements should also be considered in subsequent visits in older people to exclude orthostatic hypotension.Older people should measure their BP in upper arms by validated devices with a well readable display.In the new guidelines more attention is directed on frailty, concomitant treatments and independence influencing the likely tolerability of BP-lowering medications. In fit older patients, it is now recommended that systolic BP should be targeted to a BP range of 130 - 139 mmHg, if tolerated.Lifestyle changes are also recommended in elder patients. Preferred combination therapies are a RAS blocker with a CCB or thiazid diuretic, preferably in a "single pill". When combination therapy is used, this should be initiated at the lowest available doses in elder people.