Characterization of clumpy adhesion of Escherichia coli to human cells and associated factors influencing antibiotic sensitivity.

Escherichia coli intestinal infection pathotypes are characterized by distinct adhesion patterns, including the recently described clumpy adhesion phenotype. Here, we identify and characterize the genetic factors contributing to the clumpy adhesion of E. coli strain 4972. In this strain, the transcriptome and proteome of adhered bacteria were found to be distinct from planktonic bacteria in the supernatant. A total of 622 genes in the transcriptome were differentially expressed in bacteria present in clumps relative to the planktonic bacteria. Seven genes targeted for disruption had variable distribution in different pathotypes and nonpathogenic E. coli, with the pilV and spnT genes being the least frequent or absent from most groups. Deletion (Δ) of five differentially expressed genes, flgH, ffp, pilV, spnT, and yggT, affected motility, adhesion, or antibiotic stress. ΔflgH exhibited 80% decrease and ΔyggT depicted 184% increase in adhesion, and upon complementation, adhesion was significantly reduced to 13%. ΔflgH lost motility and was regenerated when complemented, whereas Δffp had significantly increased motility, and reintroduction of the same gene reduced it to the wild-type level. The clumps produced by Δffp and ΔspnT were more resistant and protected the bacteria, with ΔspnT showing the best clump formation in terms of ampicillin stress protection. ΔyggT had the lowest tolerance to gentamicin, where the antibiotic stress completely eliminated the bacteria. Overall, we were able to investigate the influence of clump formation on cell surface adhesion and antimicrobial tolerance, with the contribution of several factors crucial to clump formation on susceptibility to the selected antibiotics. IMPORTANCE: The study explores a biofilm-like clumpy adhesion phenotype in Escherichia coli, along with various factors and implications for antibiotic susceptibility. The phenotype permitted the bacteria to survive the onslaught of high antibiotic concentrations. Profiles of the transcriptome and proteome allowed the differentiation between adhered bacteria in clumps and planktonic bacteria in the supernatant. The deletion mutants of genes differentially expressed between adhered and planktonic bacteria, i.e., flgH, ffp, pilV, spnT, and yggT, and respective complementations in trans cemented their roles in multiple capacities. ffp, an uncharacterized gene, is involved in motility and resistance to ampicillin in a clumpy state. The work also affirms for the first time the role of the yggT gene in adhesion and its involvement in susceptibility against another aminoglycoside antibiotic, i.e., gentamicin. Overall, the study contributes to the mechanisms of biofilm-like adhesion phenotype and understanding of the antimicrobial therapy failures and infections of E. coli.

Bilateral deficiency of Meissner corpuscles and papillary microvessels in patients with acute complex regional pain syndrome.

ABSTRACT: Complex regional pain syndrome (CRPS) presents postinjury with disproportionate pain and neuropathic, autonomic, motor symptoms, and skin texture affection. However, the origin of these multiplex changes is unclear. Skin biopsies offer a window to analyze the somatosensory and vascular system as well as skin trophicity with their protecting barriers. In previous studies, barrier-protective exosomal microRNAs were altered in CRPS. We here postulated that tissue architecture and barrier proteins are already altered at the beginning of CRPS. We analyzed ipsilateral and contralateral skin biopsies of 20 fully phenotyped early CRPS patients compared with 20 age- and sex-matched healthy controls. We established several automated unbiased methods to comprehensively analyze microvessels and somatosensory receptors as well as barrier proteins, including claudin-1, claudin-5, and claudin-19. Meissner corpuscles in the skin were bilaterally reduced in acute CRPS patients with some of them lacking these completely. The number of Merkel cells and the intraepidermal nerve fiber density were not different between the groups. Dermal papillary microvessels were bilaterally less abundant in CRPS, especially in patients with allodynia. Barrier proteins in keratinocytes, perineurium of dermal nerves, Schwann cells, and papillary microvessels were not affected in early CRPS. Bilateral changes in the tissue architecture in early CRPS might indicate a predisposition for CRPS that manifests after injury. Further studies should evaluate whether these changes might be used to identify risk patients for CRPS after trauma and as biomarkers for outcome.

Transient immune activation without loss of intraepidermal innervation and associated Schwann cells in patients with complex regional pain syndrome.

BACKGROUND: Complex regional pain syndrome (CRPS) develops after injury and is characterized by disproportionate pain, oedema, and functional loss. CRPS has clinical signs of neuropathy as well as neurogenic inflammation. Here, we asked whether skin biopsies could be used to differentiate the contribution of these two systems to ultimately guide therapy. To this end, the cutaneous sensory system including nerve fibres and the recently described nociceptive Schwann cells as well as the cutaneous immune system were analysed. METHODS: We systematically deep-phenotyped CRPS patients and immunolabelled glabrous skin biopsies from the affected ipsilateral and non-affected contralateral finger of 19 acute (< 12 months) and 6 chronic (> 12 months after trauma) CRPS patients as well as 25 sex- and age-matched healthy controls (HC). Murine foot pads harvested one week after sham or chronic constriction injury were immunolabelled to assess intraepidermal Schwann cells. RESULTS: Intraepidermal Schwann cells were detected in human skin of the finger-but their density was much lower compared to mice. Acute and chronic CRPS patients suffered from moderate to severe CRPS symptoms and corresponding pain. Most patients had CRPS type I in the warm category. Their cutaneous neuroglial complex was completely unaffected despite sensory plus signs, e.g. allodynia and hyperalgesia. Cutaneous innate sentinel immune cells, e.g. mast cells and Langerhans cells, infiltrated or proliferated ipsilaterally independently of each other-but only in acute CRPS. No additional adaptive immune cells, e.g. T cells and plasma cells, infiltrated the skin. CONCLUSIONS: Diagnostic skin punch biopsies could be used to diagnose individual pathophysiology in a very heterogenous disease like acute CRPS to guide tailored treatment in the future. Since numbers of inflammatory cells and pain did not necessarily correlate, more in-depth analysis of individual patients is necessary.

Did the prevalence of depressive symptoms change during the COVID-19 pandemic? A multilevel analysis on longitudinal data from healthcare workers.

BACKGROUND: Healthcare workers (HCW) are at high risk to develop mental health problems during the COVID-19 pandemic because of additional work load, perceived stress, and exposure to patients with COVID-19. Currently, there are few studies on change over time in the prevalence of depressive symptoms during pandemic start among HCW. Thus, the aims of the current study were to examine whether depressive symptoms increased during the pandemic and were associated with perceived stress and own COVID-19 infection and workplace exposure to virus-infected patients. METHODS: The cohort study used longitudinal data from HCW collected monthly (July 2020 till December 2020) during the first year of the pandemic before vaccination became available. The sample of n = 166 was drawn from a German hospital and included medical (e.g. nurses, therapists, and physicians) and administrative staff. Using multilevel models, we analyzed the change in depressive symptoms [assessed with General Depression Scale (GDS), a validated German version of the Center for Epidemiological Studies Depression Scale (CES-D)] and its association with perceived stress across the study period. Laboratory-confirmed own infection was tested as a potential moderator in this context. Subscales of the GDS were used to examine change over time of depressive symptom modalities (e.g. emotional, somatic, and social interactions (ß, 95% confidence interval). RESULTS: Depression scores increased significantly during the study period (ß = .03, 95% CI [0.02, 0.05]). Perceived stress was associated with depressive symptoms (ß = .12, 95% CI [0.10, 0.14]) but did not change over time. Exposure to COVID-19 infection was associated with a higher increase of depressive symptoms (ß = .12, 95% CI [0.10, .14]). Somatic symptoms of depression increased among medical HCW with workplace exposure to COVID-19 (ß = .25, 95% CI [0.13, 0.38]), but not in administrators (ß = .03, 95% CI [-0.04, 0.11]). CONCLUSION: Research is needed to identify factors that promote the reduction of depressive symptoms in medical HCW with exposition to COVID-19 patients. Awareness of infection protection measures should be increased.

Markers of oxidative stress during post-COVID-19 fatigue: a hypothesis-generating, exploratory pilot study on hospital employees.

Introduction: Post-COVID-19 fatigue is common after recovery from COVID-19. Excess formation of reactive oxygen species (ROS) leading to oxidative stress-related mitochondrial dysfunction is referred to as a cause of these chronic fatigue-like symptoms. The present observational pilot study aimed to investigate a possible relationship between the course of ROS formation, subsequent oxidative stress, and post-COVID-19 fatigue. Method: A total of 21 post-COVID-19 employees of the General Hospital Nuremberg suffering from fatigue-like symptoms were studied during their first consultation (T1: on average 3 months after recovery from COVID-19), which comprised an educational talk on post-COVID-19 symptomatology and individualized outpatient strategies to resume normal activity, and 8 weeks thereafter (T2). Fatigue severity was quantified using the Chalder Fatigue Scale together with a health survey (Patient Health Questionnaire) and self-report on wellbeing (12-Item Short-Form Health Survey). We measured whole blood superoxide anion (O2•-) production rate (electron spin resonance, as a surrogate for ROS production) and oxidative stress-induced DNA strand breaks (single cell gel electrophoresis: "tail moment" in the "comet assay"). Results: Data are presented as mean ± SD or median (interquartile range) depending on the data distribution. Differences between T1 and T2 were tested using a paired Wilcoxon rank sign or t-test. Fatigue intensity decreased from 24 ± 5 at T1 to 18 ± 8 at T2 (p < 0.05), which coincided with reduced O2•- formation (from 239 ± 55 to 195 ± 59 nmol/s; p < 0.05) and attenuated DNA damage [tail moment from 0.67 (0.36-1.28) to 0.32 (0.23-0.71); p = 0.05]. Discussion: Our pilot study shows that post-COVID-19 fatigue coincides with (i) enhanced O2•- formation and oxidative stress, which are (ii) reduced with attenuation of fatigue symptoms.

Lessons learned in translating pain knowledge into practice.

Introduction: During the past 2 decades, basic research deciphering the underlying mechanisms of nociception and chronic pain was thought to finally step beyond opioids and nonsteroidals and provide patients with new analgesics. But apart from calcitonin gene-related peptide antagonists, nothing arrived in hands of clinicians. Objectives: To present existing evidence of 3 representative target molecules in the development of novel pain treatment that, so far, did not result in approved drugs. Methods: This Clinical Update aligns with the 2022 IASP Global Year Translating Pain Knowledge into Practice and selectively reviews best available evidence and practice. Results: We highlight 3 targets: a ion channel, a neuronal growth factor, and a neuropeptide to explore why these drug targets have been dropped in clinical phase II-III trials. Antibodies to nerve growth factor had very good effects in musculoskeletal pain but resulted into more patients requiring joint replacements. Blockers of NaV1.7 were often not effective enough-at least if patients were not stratified. Blockers of neurokinin receptor were similarly not successful enough. In general, failure was most often to the result of a lack of effect and to a lesser extend because of unexpected severe side effects. However, all studies and trials lead to an enormous move in the scientific community to better preclinical models and testing as well as revised methods to molecularly phenotype and stratify patients. Conclusion: All stakeholders in the process can help in the future: better preclinical studies, phenotyping and stratifying patients, and participation in clinical trials to move the discovery of analgesics forward.

Transient hypoalgesia after COVID-19 infection.

Introduction: Loss of smell or taste are often-cited complications during COVID-19 disease, but there is no clear evidence for affection of the peripheral nervous system. Methods: Here, we report a 48-year-old man presenting with persistent dysgeusia and hypoalgesia of the lower legs, hands, and cheeks after COVID-19 infection in Spring 2020. Results: Upon clinical examination 7 months after the infection, the patient could not feel pain after pinprick stimuli. Quantitative sensory testing revealed increased thermal detection thresholds at the face but no changes at the foot. Electrical C-fiber stimulation elicited lower pain ratings at the distal leg compared with the proximal leg, but overall higher pain ratings than in healthy control subjects. The axon flare reaction in response to histamine and acetylcholine was almost absent with no pain sensation. Skin punch biopsy revealed a reduced intraepidermal nerve fiber density at the lower leg, and transient receptor potential vanilloid 1 and calcitonin gene-related peptide immunoreactivity were similar to a healthy control. Symptoms and positive tests improved 5 months later. Conclusion: In summary, we describe a case of hypoalgesia after COVID-19 disease. Studies investigating long-COVID syndrome should test not only for painful neuropathic symptoms but also for hypoalgesia, especially in patients with prolonged dysgeusia.

FDG PET Hybrid Imaging.

Molecular imaging with positron emission tomography (PET) using tumour-seeking radiopharmaceuticals has gained wide acceptance in oncology with many clinical applications. The hybrid imaging modality PET/CT (computed tomography) allows assessing molecular as well as morphologic information at the same time. Therefore, PET/CT represents an efficient tool for whole-body staging and re-staging within one imaging modality. In oncology, the glucose analogue 18-F-fluorodeoxyglucose (FDG) is the most widely used PET/CT radiopharmaceutical in clinical routine. FDG PET and FDG PET/CT have been used for staging and re-staging of tumour patients in numerous studies. This chapter will discuss the use and the main indications of FDG PET/CT in oncology with special emphasis on lung cancer, lymphoma, head and neck cancer, melanoma and breast cancer (among other tumour entities). A review of the current literature is given with respect to primary diagnosis, staging and diagnosis of recurrent disease. Besides its integral role in diagnosis, staging and re-staging of disease in oncology, there is increasing evidence that FDG PET/CT can be used for therapy response assessment (possibly influencing therapeutic management and treatment planning) by evaluating tumour control, which will also be discussed in this chapter.

Stereotactically Injected Kv1.2 and CASPR2 Antisera Cause Differential Effects on CA1 Synaptic and Cellular Excitability, but Both Enhance the Vulnerability to Pro-epileptic Conditions.

PURPOSE: We present a case of voltage-gated potassium channel (VGKC) complex antibody-positive limbic encephalitis (LE) harboring autoantibodies against Kv1.2. Since the patient responded well to immunotherapy, the autoantibodies were regarded as pathogenic. We aimed to characterize the pathophysiological role of this antibody in comparison to an antibody against the VGKC-associated protein contactin-associated protein-2 (CASPR2). METHODS: Stereotactic injection of patient sera (anti-Kv1.2-associated LE or anti-CASPR2 encephalopathy) and a control subject was performed into the hippocampus of the anesthetized rat in vivo, and hippocampal slices were prepared for electrophysiological purposes. Using extra- and intracellular techniques, synaptic transmission, long-term potentiation (LTP) and vulnerability to pro-epileptic conditions were analyzed. RESULTS: We observed that the slope of the field excitatory postsynaptic potential (fEPSP) was significantly increased at Schaffer collateral-CA1 synapses in anti-Kv1.2-treated and anti-CASPR2-treated rats, but not at medial perforant path-dentate gyrus synapses. The increase of the fEPSP slope in CA1 was accompanied by a decrease of the paired-pulse ratio in anti-Kv1.2, but not in anti-CASPR2 tissue, indicating presynaptic site of anti-Kv1.2. In addition, anti-Kv1.2 tissue showed enhanced LTP in CA1, but dentate gyrus LTP remained unaltered. Importantly, LTP in slices from anti-CASPR2-treated animals did not differ from control values. Intracellular recordings from CA1 neurons revealed that the resting membrane potential and a single action potential were not different between anti-Kv1.2 and control tissue. However, when the depolarization was prolonged, the number of action potentials elicited was reduced in anti-Kv1.2-treated tissue compared to both control and anti-CASPR2 tissue. In contrast, polyspike discharges induced by removal of Mg2+ occurred earlier and more frequently in both patient sera compared to control. CONCLUSION: Patient serum containing anti-Kv1.2 facilitates presynaptic transmitter release as well as postsynaptic depolarization at the Schaffer-collateral-CA1 synapse, but not in the dentate gyrus. As a consequence, both synaptic transmission and LTP in CA1 are facilitated and action potential firing is altered. In contrast, anti-CASPR2 leads to increased postsynaptic potentials, but without changing LTP or firing properties suggesting that anti-Kv1.2 and anti-CASPR2 differ in their cellular effects. Both patient sera alter susceptibility to epileptic conditions, but presumably by different mechanisms.

Referral pathways and outcome of patients with colorectal peritoneal metastasis (CRPM).

INTRODUCTION: Traditionally patients with colorectal peritoneal metastases (CRPM) were offered palliative chemotherapy and best supportive care. With the introduction of cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC), patients in the UK have been referred to nationally approved centres. This study describes the pattern of referral and outcomes of patients managed through one UK centre. METHODS: and Methods: A prospective register recorded referrals, demographics, prior treatment pathways, and specialist multidisciplinary team (MDT) decisions (2002-2015). Peritoneal cancer index (PCI) was recorded intra-operatively; complete cytoreduction was deemed when a CC0/1 was achieved. Complications were classified using NCI CTCAE. v.4. Median overall survivals (OS) were described for those treated by CRS/HIPEC and in derived estimates for patients with isolated peritoneal metastases treated by chemotherapy alone in the ARCAD trials consortium. RESULTS: Two-hundred-eighty-six patients with CRPM were referred. Despite increasing numbers of referrals annually, the proportion of patients selected for CRS/HIPEC decreased from 64.5%, to 40%, and to 37.1% for 2002-09, 2010-12, and 2013-15, respectively (p < 0.017). CRS/HIPEC was undertaken in 117 patients with a median PCI of 7 and CC0/1 achieved in 86.3%. NCI CTCAE grade 3/4 complication rates were 9.4%; 30-day mortality was 0.85%. Median OS following CRS/HIPEC was 46.0 months: that for patients not receiving CRS/HIPEC was 13.2 months. CONCLUSION: The evolution of the national peritoneal treatment centre over 14 years has been associated with increased referral numbers, refinement of selection for major surgery, matched with achievements of low complication rates and survival advantages in selected patients compared with traditional non-surgical treatments.

Site-specific one-pot triple click labeling for DNA and RNA.

We report site-specific triple click labeling for DNA and RNA in a one-pot setup by performing inverse electron demand Diels-Alder reaction and strain-promoted and copper catalyzed click reactions sequentially. Our methodology can be applied to long and short nucleic acids alike, and could have broader potential for labeling other (bio-)molecules.

Awake Craniotomy for Tumor Resection: Further Optimizing Therapy of Brain Tumors.

In recent years more and more data have emerged linking the most radical resection to prolonged survival in patients harboring brain tumors. Since total tumor resection could increase postoperative morbidity, many methods have been suggested to reduce the risk of postoperative neurological deficits: awake craniotomy with the possibility of continuous patient-surgeon communication is one of the possibilities of finding out how radical a tumor resection can possibly be without causing permanent harm to the patient.In 1994 we started to perform awake craniotomy for glioma resection. In 2005 the use of intraoperative high-field magnetic resonance imaging (MRI) was included in the standard tumor therapy protocol. Here we review our experience in performing awake surgery for gliomas, gained in 219 patients.Patient selection by the operating surgeon and a neuropsychologist is of primary importance: the patient should feel as if they are part of the surgical team fighting against the tumor. The patient will undergo extensive neuropsychological testing, functional MRI, and fiber tractography in order to define the relationship between the tumor and the functionally relevant brain areas. Attention needs to be given at which particular time during surgery the intraoperative MRI is performed. Results from part of our series (without and with ioMRI scan) are presented.

Symmetry of the arcuate fasciculus and its impact on language performance of patients with brain tumors in the language-dominant hemisphere.

OBJECTIVE Cerebral damage in frontal, parietal, and temporal brain areas and, probably more importantly, their interconnections can lead to deficits in language. However, neural plasticity and repair allow the brain to partly compensate for neural injury, mediated by both functional and structural changes. In this study, the authors sought to systematically investigate the relationship between language performance in brain tumor patients and structural perisylvian pathways (i.e., the arcuate fasciculus [AF]) using probabilistic fiber tracking on diffusion tensor imaging. The authors used a previously proposed model in which the AF is divided into anterior, long, and posterior segments. The authors hypothesized that right-handed patients with gliomas in the language-dominant (left) hemisphere would benefit from a more symmetrical or right-lateralized language pathway in terms of better preservation of language abilities. Furthermore, they investigated to what extent specific tumor characteristics, including proximity to the AF, affect language outcome in such patients. METHODS Twenty-seven right-handed patients (12 males and 15 females; mean age 52 ± 16 years) with 11 low-grade and 16 high-grade gliomas of the left hemisphere underwent 3-T diffusion-weighted MRI (30 directions) and language assessment as part of presurgical planning. For a systematic quantitative evaluation of the AF, probabilistic fiber tracking with a 2 regions of interest approach was carried out. Volumes of the 3 segments of both hemispheric AFs were evaluated by quantifying normalized and thresholded pathways. Resulting values served to generate the laterality index of the AFs. RESULTS Patients without language deficits tended to have an AF that was symmetric or lateralized to the right, whereas patients with deficits in language significantly more often demonstrated a left-lateralized posterior segment of the AF. Patients with high-grade gliomas had more severe language deficits than those with low-grade gliomas. Backward logistic regression revealed the laterality index of the posterior AF segment and tumor grade as the only independent statistically significant predictors for language deficits in this cohort. CONCLUSIONS In addition to the well-known fact that tumor entity influences behavioral outcome, the authors' findings suggest that the right homologs of structural language-associated pathways could be supportive for language function and facilitate compensation mechanisms after brain damage in functionally eloquent areas. This further indicates that knowledge about preoperative functional redistribution (identified by neurofunctional imaging) increases the chance for total or near-total resections of tumors in eloquent areas. In the future, longitudinal studies with larger groups are mandatory to overcome the methodological limitations of this cross-sectional study and to map neuroplastic changes associated with language performance and rehabilitation in brain tumor patients.

The sensory-motor profile awake-A new tool for pre-, intra-, and postoperative assessment of sensory-motor function.

OBJECTIVES: Awake craniotomy is a well-established procedure in surgery of intracranial tumors in eloquent areas. However, sufficiently standardized instruments for the assessment of sensory-motor function before, during and after the operation are currently lacking, despite their importance for evaluation of operative outcome. PATIENTS AND METHODS: To address this issue, we designed a standardized assessment tool (the "sensory-motor profile awake scale"; SMP-a). The final scale consists of three motor sections (face, arm and leg) assessing both gross and fine motor skills and one sensory section. It differentiates between six grades of impairment and its tasks are applicable for intraoperative continuous monitoring of sensory-motor functions and supporting processes. We analyzed the data of 17 patients with intracranial tumors eligible for awake craniotomy who were preoperatively assessed with the SMP-a. In addition, we present an exemplary case. RESULTS: Our data support the assumption that the SMP-a is feasible in patients eligible for awake craniotomy, even in patients with symptoms of mild aphasia or more severe sensory-motor deficits caused by tumor recurrence. The exemplary case demonstrates the feasibility of repeated measures with the SMP-a in a tumor patient, including the adaption of tasks to the individual requirements of an intraoperative setting. CONCLUSION: This exploratory study suggests that the SMP-a might be a feasible rating scale in patients with intracranial tumors. The flexibility of the scale enables individual adaption, but preserves the standardized scoring system to allow comparison between assessment dates, patients and, hopefully in the future, institutions. However, future studies are mandatory to provide data on the instrument's diagnostic properties with respect to feasibility, objectivity, validity and reliability.

Glioblastoma in hemihydranencephaly: preoperative and postoperative language ability of the right hemisphere.

We present the first case report describing a craniotomy for a glioblastoma in a patient with hemihydranencephaly (HHE). Ten years ago our patient had average cognitive and language functions, indicating very good adaption of his single right hemisphere. Due to the tumour he developed a deceleration, deficits in language functions and mild impairments of basic cognitive functions. Further neuroplastic reorganisations of his right hemisphere in response to the tumour growth are discussed. The favourable postoperative outcome supported the decision for careful tumour resection in this patient with HHE.

Cognitive screening in patients with intracranial tumors: validation of the BCSE.

This study presents the first validation of the Brief Cognitive Status Exam (BCSE) against two other screening tools for cognitive impairment in patients with intracranial tumors. 58 patients and 22 matched healthy controls completed the BCSE, the Mini Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA). Patients were additionally tested with a comprehensive neuropsychological battery. Based on this assessment, they were classified as cognitively impaired or unimpaired on five cognitive domains. Analyses revealed a comparable feasibility of the BCSE relative to the MoCA and the MMSE, but a smaller range of assessed functions (e.g., no correlation with the domain visual-spatial functions). The ability to separate patients and healthy controls was extremely poor for BCSE and MMSE (sensitivity of 38.6 % and less), but moderate for MoCA (sensitivity 68.97 %). Detection of cognitive impairment in patients was worst with BCSE (sensitivity 37 %; MoCA 92.9 %, MMSE 44.4 %) as compared to neuropsychological testing. Moreover, prediction of cognitive outcome was also worst for the BCSE (AUC = .713, NPV = 50 %). An optimal cut-off of 50.5 increased the results slightly. In summary, the BCSE showed good feasibility but no sufficient results in separating healthy individuals from patients or detecting cognitive impairment in patients. Consequently, as a screening measure, we would recommend the MoCA instead of the BCSE. However, since even the MoCA failed to detect cognitive impairment, our study supports the view that reliable results could only be obtained with a comprehensive neuropsychological battery.

Nucleotidyl transferase assisted DNA labeling with different click chemistries.

Here, we present a simple, modular and efficient strategy that allows the 3'-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3'-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.

RNA-peptide conjugate synthesis by inverse-electron demand Diels-Alder reaction.

Here we report an efficient method for the synthesis of RNA-peptide conjugates by inverse-electron demand Diels-Alder reaction. Various dienophiles were enzymatically incorporated into RNA and reacted with a chemically synthesized diene-modified peptide. The Diels-Alder reaction proceeds with near-quantitative yields in aqueous solution with stoichiometric amounts of reactants, even at low micromolar concentrations.