Correlation of procalcitonin and c-reactive protein levels with pathogen distribution and infection localization in urinary tract infections.

Aimed to explore the relationships between infection localization, bacterial species, and procalcitonin (PCT) and C-reactive protein (CRP) levels in urinary tract infections (UTIs). A retrospective study included 314 UTI hospitalized patients divided into two groups (268 with lower UTI, 46 with upper UTI) in a tertiary care hospital. PCT and CRP were performed. Bacterial isolates were identified using standard microbiological techniques, and statistical analyses were performed to assess associations between infection localization, bacterial species, PCT, and CRP levels. Age and gender showed no significant differences between the lower and upper UTIs. Escherichia coli dominated as the leading UTI pathogen. A positive correlation (r = 0.646, P < 0.001) between PCT and CRP levels was found. The subgroup with ureteritis in the upper UTI category exhibited the highest PCT and CRP levels. PCT and CRP exhibited favorable diagnostic potential in predicting upper UTIs, with AUCs of 0.644 and 0.629, respectively. The optimal cutoff values were 0.21 ng/mL for PCT and 60.77 mg/L for CRP. Sensitivities were 69.03% and 77.99%, while specificities were 56.52% and 47.83%, respectively. E. coli emerged as the predominant bacterium in UTIs. PCT and CRP demonstrated moderate diagnostic efficacy in distinguishing between upper and lower UTIs. Notably, PCT and CRP exhibited enhanced utility in identifying ureteritis.

Impact of Biometric Patient Data, Probiotic Supplementation, and Selected Gut Microorganisms on Calprotectin, Zonulin, and sIgA Concentrations in the Stool of Adults Aged 18-74 Years.

Alterations to the intestinal barrier may be involved in the pathogenesis of various chronic diseases. The diagnosis of mucosal barrier disruption has become a new therapeutic target for disease prevention. The aim of this study was to determine whether various patient demographic and biometric data, often not included in diagnostic analyses, may affect calprotectin, zonulin, and sIgA biomarker values. Stool markers' levels in 160 samples were measured colorimetrically. The analysis of twenty key bacteria (15 genera and 5 species) was carried out on the basis of diagnostic tests, including cultures and molecular tests. The concentrations of selected markers were within reference ranges for most patients. The sIgA level was significantly lower in participants declaring probiotics supplementation (p = 0.0464). We did not observe differences in gastrointestinal discomfort in participants. We found significant differences in the sIgA level between the 29-55 years and >55 years age-related intervals groups (p = 0.0191), together with a significant decreasing trend (p = 0.0337) in age-dependent sIgA concentration. We observed complex interdependencies and relationships between their microbiota and the analyzed biomarkers. For correct clinical application, standardized values of calprotectin and sIgA should be determined, especially in elderly patients. We observed a correlation between the composition of the gut community and biomarker levels, although it requires further in-depth analysis.

Development of a selective, sensitive and robust oxyntomodulin dual monoclonal antibody immunoassay.

Background & Aim: Oxyntomodulin (Oxm) is a proglucagon-derived peptide agonist of both the GLP-1 and glucagon receptors and is a key regulator of gastric acid secretion and energy expenditure. Differential processing from proglucagon hinders assay immunoassay selectivity. Method & results: Antibody engineering was used to develop a sandwich immunoassay that selectively measures endogenous Oxm. The pre- and postprandial levels of Oxm from 19 healthy individuals over the course of 2 h were measured. Postprandial increases in Oxm occurred within minutes and levels significantly correlated with those obtained using previously published mass spectrometry assays. Conclusion: This sandwich immunoassay is appropriately sensitive and selective and is also amenable to high-throughput application for the reliable determination of endogenous levels of intact Oxm from human samples.

Mid-Regional Pro-Adrenomedullin in Combination With Pediatric Early Warning Scores for Risk Stratification of Febrile Children Presenting to the Emergency Department: Secondary Analysis of a Nonprespecified United Kingdom Cohort Study.

OBJECTIVES: Current sepsis guidelines do not provide good risk stratification of subgroups in whom prompt IV antibiotics and fluid resuscitation might of benefit. We evaluated the utility of mid-regional pro-adrenomedullin (MR-proADM) in identification of patient subgroups at risk of requiring PICU or high-dependency unit (HDU) admission or fluid resuscitation. DESIGN: Secondary, nonprespecified analysis of prospectively collected dataset. SETTING: Pediatric Emergency Department in a United Kingdom tertiary center. PATIENTS: Children less than 16 years old presenting with fever and clinical indication for venous blood sampling ( n = 1,183). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Primary outcome measures were PICU/HDU admission or administration of fluid resuscitation, with a secondary outcome of definite or probable bacterial infection. Biomarkers were measured on stored plasma samples and children phenotyped into bacterial and viral groups using a previously published algorithm. Of the 1,183 cases, 146 children (12.3%) required fluids, 48 (4.1%) were admitted to the PICU/HDU, and 244 (20.6%) had definite or probable bacterial infection. Area under the receiver operating characteristic (AUC) was used to assess performance. MR-proADM better predicted fluid resuscitation (AUC, 0.73; 95% CI, 0.67-0.78), than both procalcitonin (AUC, 0.65; 95% CI, 0.59-0.71) and Pediatric Early Warning Score (PEWS: AUC, 0.62; 95% CI, 0.56-0.67). PEWS alone showed good accuracy for PICU/HDU admission 0.83 (0.78-0.89). Patient subgroups with high MR-proADM (≥ 0.7 nmol/L) and high procalcitonin (≥ 0.5 ng/mL) had increased association with PICU/HDU admission, fluid resuscitation, and bacterial infection compared with subgroups with low MR-proADM (< 0.7 nmol/L). For children with procalcitonin less than 0.5 ng/mL, high MR-proADM improved stratification for fluid resuscitation only. CONCLUSIONS: High MR-proADM and high procalcitonin were associated with increased likelihood of subsequent disease progression. Incorporating MR-proADM into clinical risk stratification may be useful in clinician decision-making regarding initiation of IV antibiotics, fluid resuscitation, and escalation to PICU/HDU admission.

Immunohistochemical findings in the lungs of COVID-19 subjects: evidence of surfactant dysregulation.

OBJECTIVE: Acute respiratory distress syndrome (ARDS) is characterized by quantitative and qualitative changes in surfactant composition, leading to surfactant dysregulation with alveolar collapse and acute respiratory hypoxic failure. Recently, surfactant has been hypothesized to play a relevant role in COVID-19, representing a strong defender against SARS-CoV-2 infection. The aim of our work was the study of immunohistochemical surfactant expression in the lungs of patients died following SARS-CoV-2 ARDS, in order to shed light on a possible therapeutic surfactant administration. PATIENTS AND METHODS: We investigated four patients who died due to ARDS following SARS-COV-2 infection and four patients submitted to lung biopsy, in the absence of SARS-CoV-2 infection. In all 8 cases, lung specimens were immunostained with anti-surfactant protein A (SP-A) and B (SP-B). RESULTS: In control subjects, reactivity for SP-B was restricted to type II alveolar cells. Immunostaining for SP-A was observed on the surface of alveolar spaces. In the COVID-19 positive lungs, immunoreactivity for SP-B was similar to that observed in control lungs; SP-A was strongly expressed along the alveolar wall. Moreover, dense aggregates of SP-A positive material were observed in the alveolar spaces. CONCLUSIONS: Our immunohistochemical data show the dysregulation of surfactant production in COVID-19 patients, particularly regarding SP-A expression. The increased presence of SP-A in condensed masses inside alveolar spaces could invalidate the therapeutic efficacy of the treatment with exogenous surfactant.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

Association of endothelial activation assessed through endothelin-I precursor peptide measurement with mortality in COVID-19 patients: an observational analysis.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) has been linked to thrombotic complications and endothelial dysfunction. We assessed the prognostic implications of endothelial activation through measurement of endothelin-I precursor peptide (proET-1), the stable precursor protein of Endothelin-1, in a well-defined cohort of patients hospitalized with COVID-19. METHODS: We measured proET-1 in 74 consecutively admitted adult patients with confirmed COVID-19 and compared its prognostic accuracy to that of patients with community-acquired pneumonia (n = 876) and viral bronchitis (n = 371) from a previous study by means of logistic regression analysis. The primary endpoint was all-cause 30-day mortality. RESULTS: Overall, median admission proET-1 levels were lower in COVID-19 patients compared to those with pneumonia and exacerbated bronchitis, respectively (57.0 pmol/l vs. 113.0 pmol/l vs. 96.0 pmol/l, p < 0.01). Although COVID-19 non-survivors had 1.5-fold higher admission proET-1 levels compared to survivors (81.8 pmol/l [IQR: 76 to 118] vs. 53.6 [IQR: 37 to 69]), no significant association of proET-1 levels and mortality was found in a regression model adjusted for age, gender, creatinine level, diastolic blood pressure as well as cancer and coronary artery disease (adjusted OR 0.1, 95% CI 0.0009 to 14.7). In patients with pneumonia (adjusted OR 25.4, 95% CI 5.1 to 127.4) and exacerbated bronchitis (adjusted OR 120.1, 95% CI 1.9 to 7499) we found significant associations of proET-1 and mortality. CONCLUSIONS: Compared to other types of pulmonary infection, COVID-19 shows only a mild activation of the endothelium as assessed through measurement of proET-1. Therefore, the high mortality associated with COVID-19 may not be attributed to endothelial dysfunction by the surrogate marker proET-1.

Targeted Top-Down Mass Spectrometry for the Characterization and Tissue-Specific Functional Discovery of Crustacean Hyperglycemic Hormones (CHH) and CHH Precursor-Related Peptides in Response to Low pH Stress.

Crustacean hyperglycemic hormones (CHHs) are a family of neuropeptides that were discovered in multiple tissues in crustaceans, but the function of most isoforms remains unclear. Functional discovery often requires comprehensive qualitative profiling and quantitative analysis. The conventional enzymatic digestion method has several limitations, such as missing post-translational modification (PTM) information, homology interference, and incomplete sequence coverage. Herein, by using a targeted top-down method, facilitated by higher sensitivity instruments and hybrid fragmentation modes, we achieved the characterization of two CHH isoforms from the sinus glands (SG-CHH) and the pericardial organs (PO-CHH) from the Atlantic blue crab, Callinectes sapidus, with improved sequence coverage compared to earlier studies. In this study, both label-free and isotopic labeling approaches were adopted to monitor the response of CHHs and CHH precursor-related peptide (CPRP) under low pH stress. The identical trends of CPRP and CHH expression indicated that CPRP could serve as an ideal probe in tracking the CHH expression level changes, which would greatly simplify the quantitative analysis of large peptides. Furthermore, the distinct patterns of changes in the expression of CHHs in the SG and the PO suggested their tissue-specific functions in the regulation of low pH stress. Ion mobility-mass spectrometry (IM-MS) was also employed in this study to provide conformation analysis of both CHHs and CPRPs from different tissues.

Efferent projections of Vglut2, Foxp2, and Pdyn parabrachial neurons in mice.

The parabrachial nucleus (PB) is a complex structure located at the junction of the midbrain and hindbrain. Its neurons have diverse genetic profiles and influence a variety of homeostatic functions. While its cytoarchitecture and overall efferent projections are known, we lack comprehensive information on the projection patterns of specific neuronal subtypes in the PB. In this study, we compared the projection patterns of glutamatergic neurons here with a subpopulation expressing the transcription factor Foxp2 and a further subpopulation expressing the neuropeptide Pdyn. To do this, we injected an AAV into the PB region to deliver a Cre-dependent anterograde tracer (synaptophysin-mCherry) in three different strains of Cre-driver mice. We then analyzed 147 neuroanatomical regions for labeled boutons in every brain (n = 11). Overall, glutamatergic neurons in the PB region project to a wide variety of sites in the cerebral cortex, basal forebrain, bed nucleus of the stria terminalis, amygdala, diencephalon, and brainstem. Foxp2 and Pdyn subpopulations project heavily to the hypothalamus, but not to the cortex, basal forebrain, or amygdala. Among the few differences between Foxp2 and Pdyn cases was a notable lack of Pdyn projections to the ventromedial hypothalamic nucleus. Our results indicate that genetic identity determines connectivity (and therefore, function), providing a framework for mapping all PB output projections based on the genetic identity of its neurons. Using genetic markers to systematically classify PB neurons and their efferent projections will enhance the translation of research findings from experimental animals to humans.

Input-output connections of LJA5 prodynorphin neurons.

Sensory information is transmitted from peripheral nerves, through the spinal cord, and up to the brain. Sensory information may be modulated by projections from the brain to the spinal cord, but the neural substrates for top-down sensory control are incompletely understood. We identified a novel population of inhibitory neurons in the mouse brainstem, distinguished by their expression of prodynorphin, which we named LJA5. Here, we identify a similar group of Pdyn+ neurons in the human brainstem, and we define the efferent and afferent projection patterns of LJA5 neurons in mouse. Using specific genetic tools, we selectively traced the projections of the Pdyn-expressing LJA5 neurons through the brain and spinal cord. Terminal fields were densest in the lateral and ventrolateral periaqueductal gray (PAG), lateral parabrachial nucleus (LPB), caudal pressor area, and lamina I of the spinal trigeminal nucleus and all levels of the spinal cord. We then labeled cell types in the PAG, LPB, medulla, and spinal cord to better define the specific targets of LJA5 boutons. LJA5 neurons send the only known inhibitory descending projection specifically to lamina I of the spinal cord, which transmits afferent pain, temperature, and itch information up to the brain. Using retrograde tracing, we found LJA5 neurons receive inputs from sensory and stress areas such as somatosensory/insular cortex, preoptic area, paraventricular nucleus, dorsomedial nucleus and lateral hypothalamus, PAG, and LPB. This pattern of inputs and outputs suggest LJA5 neurons are uniquely positioned to be activated by sensation and stress, and in turn, inhibit pain and itch.

Evaluation of serum zonulin level in prediabetic patients.

BACKGROUND: This study aimed to investigate the relationship between lipopolysaccharide (LPS) and zonulin levels and also to show the effect of acute hyperglycemic stress induced by oral glucose tolerance testing (OGTT) on zonulin levels in pre-diabetic patients. METHODS: Four groups were constituted according to the criteria of the American Diabetes Association (ADA), based on OGTT results: control group (n:40); prediabetic group (n:56), divided into two subgroups: impaired fasting glucose group (IFG) (n:36), and impaired glucose tolerance (IGT) + IFG group (n:20) and type-2 diabetes mellitus (T2DM) group (n:45). RESULTS: Zonulin and LPS did not significantly differ between the prediabetes and control groups, but were significantly higher in the T2DM group compared to both the prediabetic and the control group (P<0.001). After OGTT, zonulin and LPS were significantly higher in the prediabetes group compared to the control group (P<0.01 and P<0.05, respectively), and significantly lower in the IFG and IFG+IGT groups compared to the T2DM group (P<0.001, P<0.001 and P<0.001, P<0.001, respectively). A positive correlation was detected between fasting zonulin and 2-hour zonulin (r=0.727, P<0.001) and between fasting LPS (r=0.555, P<0.001) and 2-hour LPS (r=0.567, P<0.001) in the prediabetic group. Increased zonulin and LPS levels and the positive correlation between these levels during the prediabetic period although non significant suggests onset of intestinal permeability. CONCLUSIONS: During acute hyperglycemia in prediabetic patients, up-regulation of zonulin and LPS may affect intestinal function. The intestines may play a key role in up-regulation of glucose and the pathogenesis of diabetes.

Matrix-Assisted Ionization and Tandem Mass Spectrometry Capabilities in Protein Biomarker Characterization-An Initial Study Using the Small Cell Lung Cancer Biomarker Progastrin Releasing Peptide as a Model Compound.

This initial study evaluates vacuum matrix-assisted ionization (vMAI) mass spectrometry (MS) for identification and determination of tryptic peptides from the biomarker protein progastrin releasing peptide (ProGRP). Similar peptides and charge states were observed as in liquid chromatography (LC) electrospray ionization (ESI) MS. The prolonged ion duration in vMAI with similar charge states as in ESI was advantageous for determining the MS/MS fragmentation conditions compared to MAI. It is assumed that the vacuum ionization conditions lower the detection limits of the experiment. This may be the reason vMAI combined with high resolution MS enabled detection of tryptic peptides from more digested proteins than MAI selected reaction monitoring MS. Additionally, MAI ion mobility spectrometry MS (MAI-IMS-MS) was evaluated for differentiation of intact protein isoforms, successfully enabling differentiation of the isoforms by drift time selection. Examples are both shown for model proteins bovine serum albumin, cytochrome C, and lysozyme and the clinically relevant small cell lung cancer protein biomarker ProGRP, which exists in three isoforms. Coupling with the vacuum ionization conditions using a dedicated vacuum-probe source MAI enables information to be extracted readily as with conventional approaches, just faster.

Hepatitis B Virus Pre-S Mutants as Biomarkers and Targets for the Development and Recurrence of Hepatocellular Carcinoma.

Chronic hepatitis B virus (HBV) infection is a major risk factor for the development of hepatocellular carcinoma (HCC), the leading cause of cancer-related death worldwide. Despite progress in the prevention and therapy of HCC, high incidence and recurrence rates of HCC remain big threats, resulting in poor patient survival. Effective biomarkers and targets of HCC are therefore urgently needed for better management and to improve patient outcomes. Pre-S mutants have been well demonstrated as HBV oncoproteins that play important roles in HCC development through activation of multiple oncogenic signal pathways in hepatocytes, in vitro and in vivo. The presence of pre-S mutants in patients with chronic HBV infection and HBV-related HCC has been associated with a significantly higher risk of HCC development and recurrence after curative surgical resection, respectively. In this review, we summarize the roles of pre-S mutants as biomarkers for predicting HBV-related HCC development and recurrence, and highlight the pre-S mutants-activated oncogenic signal pathways as potential targets for preventing HBV-related HCC development.

Effects of Probiotic Strains on Disease Activity and Enteric Permeability in Psoriatic Arthritis-A Pilot Open-Label Study.

(1) Background: Psoriatic Arthritis (PsA) is a painful disease of the joints and spine. Recent reports observed distinct enteric dysbiosis in PsA; intake of probiotic strains is considered to ameliorate enteric dysbiosis. If probiotics are effective in PsA is elusive. (2) Methods: In this pilot open-label study we enrolled 10 PsA patients with low to medium disease activity who received probiotics for 12 weeks. Analysis of faecal zonulin, α1-antitrypsin and calprotectin, as well as peripheral immune phenotyping was performed at baseline, after 12 weeks and 12 weeks after termination of probiotic intake. (3) Results: All patients showed increased levels of the enteric permeability marker zonulin which correlated with the frequency of peripheral Th17 cells. Calprotectin, a marker for intestinal inflammation was elevated in 6 out of 10 patients. Probiotic intake resulted in a reduction of disease activity and gut permeability. These effects, however, were not sustained beyond termination of probiotic intake. (4) Conclusions: PsA patients suffer from enhanced enteric permeability and inflammation. Probiotics may ameliorate disease activity in PsA by targeting these alterations.

Protein biomarkers associated with frozen Japanese puffer fish (Takifugu rubripes) quality traits.

This study was designed to investigate proteome changes in Japanese puffer fish (Takifugu rubripes) during short- and long-term frozen storage. In total, 1484 proteins were quantified, and 164 proteins were identified as differential abundance proteins (DAPs) in Japanese puffer fish from two frozen storage treatment groups (14 days and 60 days) compared with the fresh control group. Correlation analysis between the DAPs and quality traits of the puffer fish muscle showed that 106 proteins were correlated closely with colour and texture (hardness, elasticity, and chewiness). Bioinformatics analysis revealed and Western blot analysis verified that Putative prothymosin alpha species, Bridging integrator 3, NADH: the ubiquinone oxidoreductase subunit and Mx species are candidate biomarkers for puffer fish properties. This study offers valuable evidence to improve the quality control and monitoring of Japanese puffer fish during transportation and storage.

Neonatal CSF vasopressin concentration predicts later medical record diagnoses of autism spectrum disorder.

Autism spectrum disorder (ASD) is a brain disorder characterized by social impairments. ASD is currently diagnosed on the basis of behavioral criteria because no robust biomarkers have been identified. However, we recently found that cerebrospinal fluid (CSF) concentration of the "social" neuropeptide arginine vasopressin (AVP) is significantly lower in pediatric ASD cases vs. controls. As an initial step in establishing the direction of causation for this association, we capitalized upon a rare biomaterials collection of newborn CSF samples to conduct a quasi-prospective test of whether this association held before the developmental period when ASD first manifests. CSF samples had been collected in the course of medical care of 0- to 3-mo-old febrile infants (n = 913) and subsequently archived at -70 °C. We identified a subset of CSF samples from individuals later diagnosed with ASD, matched them 1:2 with appropriate controls (n = 33 total), and quantified their AVP and oxytocin (OXT) concentrations. Neonatal CSF AVP concentrations were significantly lower among ASD cases than controls and individually predicted case status, with highest precision when cases with comorbid attention-deficit/hyperactivity disorder were removed from the analysis. The associations were specific to AVP, as ASD cases and controls did not differ in neonatal CSF concentrations of the structurally related neuropeptide, OXT. These preliminary findings suggest that a neurochemical marker of ASD may be present very early in life, and if replicated in a larger, prospective study, this approach could transform how ASD is detected, both in behaviorally symptomatic children, and in infants at risk for developing it.

A pro-gastrin-releasing peptide imprinted photoelectrochemical sensor based on the in situ growth of gold nanoparticles on a MoS2 nanosheet surface.

Lamellar MoS2 nanosheets were successfully prepared by hydrothermal synthesis using 1-(3-mercaptopropyl)-3-methyl-imidazolium bromine (MIMBr) ionic liquid as a sulfur source and a morphology control agent, and sodium molybdate as a molybdenum source. Gold nanoparticles were assembled on the surface of MoS2 nanosheets by the in situ reduction of chloroauric acid at low temperatures to successfully fabricate AuNP/2D-MoS2 nanocomposites, thus improving photoelectrochemical response. AuNP/2D-MoS2 nanocomposites were used as photoelectrically active materials modified onto a glassy carbon electrode surface to construct a photoelectrochemical (PEC) sensor. Then, using 1-(N-pyrrolpropyl)-3-methyl-imidazolium bromine (PMIMBr) ionic liquid as a functional monomer and pro-gastrin-releasing peptide (Pro-GRP) as a template, a molecularly imprinted polymerized ionic liquid film was electrochemically deposited on an AuNP/2D-MoS2/GCE surface. Upon removing the templates, a molecularly imprinted photoelectrochemical sensor was constructed for the sensing of a tumor marker, pro-gastrin-releasing peptide. Experimental conditions including ascorbic acid concentration, polymerization conditions, incubation time, and pH value of the incubation solution have been optimized. Under the optimized conditions, the molecularly imprinted photoelectrochemical sensor can specifically detect the target protein Pro-GRP in the range of 0.02 ng mL-1-5 ng mL-1 with a detection limit of 0.0032 ng mL-1 (S/N = 3). The practicability of this photoelectrochemical sensor was demonstrated by accurately determining Pro-GRP in human serum samples.

Toc75-V/OEP80 is processed during translocation into chloroplasts, and the membrane-embedded form exposes its POTRA domain to the intermembrane space.

The insertion of membrane proteins requires proteinaceous complexes in the cytoplasm, the membrane, and the lumen of organelles. Most of the required complexes have been described, while the components for insertion of ß-barrel-type proteins into the outer membrane of chloroplasts remain unknown. The same holds true for the signals required for the insertion of ß-barrel-type proteins. At present, only the processing of Toc75-III, the ß-barrel-type protein of the central chloroplast translocon with an atypical signal, has been explored in detail. However, it has been debated whether Toc75-V/ outer envelope protein 80 (OEP80), a second protein of the same family, contains a signal and undergoes processing. To substantiate the hypothesis that Toc75-V/OEP80 is processed as well, we reinvestigated the processing in a protoplast-based assay as well as in native membranes. Our results confirm the existence of a cleavable segment. By protease protection and pegylation, we observed intermembrane space localization of the soluble N-terminal domain. Thus, Toc75-V contains a cleavable N-terminal signal and exposes its polypeptide transport-associated domains to the intermembrane space of plastids, where it likely interacts with its substrates.