Single cell technologies for monitoring protein secretion heterogeneity.

Cell-to-cell heterogeneity presents challenges across various fields, from biomedicine to bioproduction, where precise cellular responses are vital. While single cell technologies have significantly enhanced our understanding of population heterogeneity, the predominant focus has been on monitoring intracellular compounds. Recognizing the added complexity introduced by the secretion system, in this review, we first provide a systematic overview of the distinct steps necessary for driving protein secretion. We discuss the various sources of noise acting from the synthesized preprotein to the secretory protein released based on a Gram-positive cellular system as a model. We next explore the applicability of single cell technologies for monitoring protein secretion throughout these functional stages. We also emphasize the importance of applying these single cell technologies for monitoring protein secretion during bioproduction.

Feasibility assessment of a low-cost visible spectroscopy-based prototype for monitoring polyphenol extraction in fermenting musts.

BACKGROUND: Polyphenols have long been used to evaluate grape and wine quality and it is necessary to measure them throughout various winemaking stages. They are currently assessed predominantly through analytical methods, which are characterized by time-consuming procedures and environmentally harmful practices. Non-destructive spectroscopy-based devices offer an alternative but they tend to be costly and not readily accessible for smaller wineries. This study introduces the initial steps in employing a portable, user-friendly, and cost-effective visible (VIS) spectrophotometer prototype for direct polyphenol measurement during winemaking. RESULTS: Grapes (cv Syrah, Bobal, and Cabernet Sauvignon) at different maturation stages were fermented with or without stems. Throughout fermentation, parameters such as color intensity, total polyphenol index, total anthocyanins, and tannins were monitored. Concurrently, VIS spectra were acquired using both the prototype and a commercial instrument. Chemometric approaches were then applied to establish correlation models between spectra and destructive analyses. The prototype models demonstrated an acceptable level of confidence for only a few parameters, indicating their lack of complete reliability at this stage. CONCLUSIONS: Visible spectroscopy is already utilized for polyphenol analysis in winemaking but the aspiration to automate the process in wineries, particularly with low-cost devices, remains unrealized. This study investigates the feasibility of a low-cost and user-friendly spectrophotometer. The results indicate that, in the early stages of prototype utilization, the goal is attainable but requires further development and in-depth assessments. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Upper Limb Orthosis in the Rehabilitation of Stroke Patients: The Role of 3D Printing.

Stroke represents the third cause of long-term disability in the world. About 80% of stroke patients have an impairment of bio-motor functions and over half fail to regain arm functionality, resulting in motor movement control disorder with serious loss in terms of social independence. Therefore, rehabilitation plays a key role in the reduction of patient disabilities, and 3D printing (3DP) has showed interesting improvements in related fields, thanks to the possibility to produce customized, eco-sustainable and cost-effective orthoses. This study investigated the clinical use of 3DP orthosis in rehabilitation compared to the traditional ones, focusing on the correlation between 3DP technology, therapy and outcomes. We screened 138 articles from PubMed, Scopus and Web of Science, selecting the 10 articles fulfilling the inclusion criteria, which were subsequently examined for the systematic review. The results showed that 3DP provides substantial advantages in terms of upper limb orthosis designed on the patient's needs. Moreover, seven research activities used biodegradable/recyclable materials, underlining the great potential of validated 3DP solutions in a clinical rehabilitation setting. The aim of this study was to highlight how 3DP could overcome the limitations of standard medical devices in order to support clinicians, bioengineers and innovation managers during the implementation of Healthcare 4.0.

A case of septic arthritis caused by Capnocytophaga canimorsus in an HIV patient.

Invasive infections caused by Capnocytophaga canimorsus , a Gram-negative rod found in the oral cavity of healthy dogs and cats, are rare but they are increasing worldwide. We report a case of septic arthritis in a native knee joint due to this micro-organism. A 57-year-old man, with a well-controlled chronic HIV infection, attended the Emergency Department because of left knee pain and shivering without measured fever. A knee arthrocentesis and a computed tomography scan were performed, revealing septic arthritis with collections in the left leg posterior musculature. He was admitted to the Infectious Diseases Department for antibiotic treatment. Initial synovial fluid was inoculated in blood culture bottles, and the anaerobic one was positive after 63 h. Gram stain revealed fusiform Gram-negative rods, identified as C. canimorsus by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) directly from the bottle. Identification was confirmed by 16S rRNA sequencing and serotyping was performed by PCR, with serovar A as the outcome. Due to an unfavourable clinical course, the patient required two surgical cleanings and after appropriate antibiotic treatment he was discharged 2 months later.

Cellulitis and transient bacteremia by Capnocytophaga canis after a cat scratch in an immunocompetent patient.

Capnocytophaga canis is still a rare cause of infection. We present a case of an immunocompetent patient admited in the hospital with functional impotence, pain and erythema in his left leg after suffering two scratches from his cat 48 h ago. After obtaining blood and wound cultures, broad-spectrum antimicrobial therapy with intravenous amoxicillin clavulanate was initiated. After 1 day and with a clear improvement of the symptoms the patient was discharged from the hospital with cellulitis and transient bacteremia as diagnosis and completing 1 week of antimicrobial therapy orally. After 80 and 92 h of incubation, both anaerobic flasks were positive. In the Gram-stain Gram-negative rod-shaped bacteria could be observed. Despite subculturing in brucella blood agar, tripticase soy agar with 5 % of sheep blood and chocolate agar, in both anaerobic and microaerophilic conditions, the strain could not be recovered. However, these Gram-negative rods could be identified as C. canis by 16S rRNA sequencing, Capsular typing was performed to study the strain, but none of the studied capsule-types tested positive. C. canis is still a rare cause of human infection, but it must be considered in the differential diagnosis of infections related to bites, scratches and licks from dogs or cats.

Sepsis por Capnocytophaga canimorsus en unpaciente inmunocompetente / Sepsis caused by Capnocytophaga canimorsus in an immunocompetent patient

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Septic shock caused by Capnocytophaga canis after a cat scratch.

Capnocytophaga canis is an uncommon cause of septic shock. Only three cases have been previously reported in the literature. In this article, we describe the case of a 70-year-old male admitted to the intensive care unit for septic shock of unknown origin. On day 2, one anaerobic bottle out of the two sets taken at admission turned positive with Gram-negative bacilli. The pathogen was identified by 16S rRNA gene as C. canis. The strain was characterized and compared with other clinical isolates of Capnocytophaga spp.

Capnocytophaga canimorsus Capsular Serovar and Disease Severity, Helsinki Hospital District, Finland, 2000-2017.

We assembled a collection of 73 Capnocytophaga canimorsus isolates obtained from blood cultures taken from patients treated at Helsinki University Hospital (Helsinki, Finland) during 2000-2017. We serotyped these isolates by PCR and Western blot and attempted to correlate pathogen serovar with patient characteristics. Our analyses showed, in agreement with previous research, that 3 C. canimorsus serovars (A-C) caused most (91.8%) human infections, despite constituting only 7.6% of isolates found in dogs. The 3 fatalities that occurred in our cohort were equally represented by these serovars. We found 2 untypeable isolates, which we designated serovars J and K. We did not detect an association between serovar and disease severity, immune status, alcohol abuse, or smoking status, but dog bites occurred more frequently among patients infected with non-A-C serovars. Future research is needed to confirm serovar virulence and develop strategies to reduce risk for these infections in humans.

Capsular serovars of virulent Capnocytophaga canimorsus are shared by the closely related species C. canis and C. cynodegmi.

Capnocytophaga canimorsus is a dog oral commensal bacterium that causes rare but life-threatening generalized infections in humans who have been in contact with its animal hosts. Two other dog commensals, Capnocytophaga canis and Capnocytophaga cynodegmi, cause rare, mild local infections. To date, nine capsular serovars have been described in C. canimorsus. Here, we serotyped 112 strains of Capnocytophaga spp. isolated from human infections. The C. canimorsus strains (86 of 96, 89.6%) belonged to serovars A, B, or C with relative frequencies of approximately 30% for each serovar. The high prevalence of the A, B, and C serovars in strains isolated from humans, compared to the previously described low prevalence of these serovars among dog isolates (7.6%), confirms that these three serovars are more virulent to humans than other serovars and suggests that the low incidence of disease may be linked to the low prevalence of the A, B, and C serovars in dogs. We serotyped six strains of C. canis and ten strains of C. cynodegmi and, surprisingly, found one C. canis and three C. cynodegmi strains to be of capsular serovar B. This observation prompted us to test 34 dog-isolated C. canis and 16 dog-isolated C. cynodegmi strains. We found four C. canis strains belonging to serovar A and one belonging to serovar F. In contrast, no dog-isolated C. cynodegmi strain could be typed with the available antisera. This work demonstrates that virulence-associated capsular polysaccharides (A, B, and C) are not specific to the C. canimorsus species.

Identification of Virulent Capnocytophaga canimorsus Isolates by Capsular Typing.

Capnocytophaga canimorsus is a dog oral commensal that causes rare but severe infections in humans. C. canimorsus was recently shown to be endowed with a capsular polysaccharide implicated in resistance to the innate immune system of the host. Here, we developed the first C. canimorsus capsular serotyping scheme. We describe nine different serovars (A to I), and this serotyping scheme allowed typing of 25/25 isolates from human infections but only 18/52 isolates from dog mouths, indicating that the repertoire of capsules in the species is vast. However, while only three serovars (A, B, and C) covered 88% of the human isolates tested (22/25), they covered only 7.7% of the dog isolates (4/52). Serovars A, B, and C were found 22.9-, 14.6-, and 4.2-fold more often, respectively, among human isolates than among dog isolates, with no geographical bias, implying that isolates endowed with these three capsular types are more virulent for humans than other isolates. Capsular serotyping would thus allow identification of virulent isolates in dogs, which could contribute to the prevention of these infections. To this end, we developed a PCR typing method based on the amplification of specific capsular genes.

Evidence for a LOS and a capsular polysaccharide in Capnocytophaga canimorsus.

Capnocytophaga canimorsus is a dog's and cat's oral commensal which can cause fatal human infections upon bites or scratches. Infections mainly start with flu-like symptoms but can rapidly evolve in fatal septicaemia with a mortality as high as 40%. Here we present the discovery of a polysaccharide capsule (CPS) at the surface of C. canimorsus 5 (Cc5), a strain isolated from a fulminant septicaemia. We provide genetic and chemical data showing that this capsule is related to the lipooligosaccharide (LOS) and probably composed of the same polysaccharide units. A CPS was also found in nine out of nine other strains of C. canimorsus. In addition, the genomes of three of these strains, sequenced previously, contain genes similar to those encoding CPS biosynthesis in Cc5. Thus, the presence of a CPS is likely to be a common property of C. canimorsus. The CPS and not the LOS confers protection against the bactericidal effect of human serum and phagocytosis by macrophages. An antiserum raised against the capsule increased the killing of C. canimorsus by human serum thus showing that anti-capsule antibodies have a protective role. These findings provide a new major element in the understanding of the pathogenesis of C. canimorsus.

Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria.

Bacteria of the phylum Bacteroidetes, including commensal organisms and opportunistic pathogens, harbor abundant surface-exposed multiprotein membrane complexes (Sus-like systems) involved in carbohydrate acquisition. These complexes have been mostly linked to commensalism, and in some instances, they have also been shown to play a role in pathogenesis. Sus-like systems are mainly composed of lipoproteins anchored to the outer membrane and facing the external milieu. This lipoprotein localization is uncommon in most studied Gram-negative bacteria, while it is widespread in Bacteroidetes Little is known about how these complexes assemble and particularly about how lipoproteins reach the bacterial surface. Here, by bioinformatic analyses, we identify a lipoprotein export signal (LES) at the N termini of surface-exposed lipoproteins of the human pathogen Capnocytophaga canimorsus corresponding to K-(D/E)2 or Q-A-(D/E)2 We show that, when introduced in sialidase SiaC, an intracellular lipoprotein, this signal is sufficient to target the protein to the cell surface. Mutational analysis of the LES in this reporter system showed that the amino acid composition, position of the signal sequence, and global charge are critical for lipoprotein surface transport. These findings were further confirmed by the analysis of the LES of mucinase MucG, a naturally surface-exposed C. canimorsus lipoprotein. Furthermore, we identify a LES in Bacteroides fragilis and Flavobacterium johnsoniae surface lipoproteins that allow C. canimorsus surface protein exposure, thus suggesting that Bacteroidetes share a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane. IMPORTANCE: Bacteria of the phylum Bacteroidetes are important human commensals and pathogens. Understanding their biology is therefore a key question for human health. A main feature of these bacteria is the presence of abundant lipoproteins at their surface that play a role in nutrient acquisition. To date, the underlying mechanism of lipoprotein transport is unknown. We show for the first time that Bacteroidetes surface lipoproteins share an N-terminal signal that drives surface localization. The localization and overall negative charge of the lipoprotein export signal (LES) are crucial for its role. Overall, our findings provide the first evidence that Bacteroidetes are endowed with a new bacterial lipoprotein export pathway that flips lipoproteins across the outer membrane.

Improvement of identification of Capnocytophaga canimorsus by matrix-assisted laser desorption ionization-time of flight mass spectrometry using enriched database.

Capnocytophaga canimorsus and Capnocytophaga cynodegmi can be transmitted from dogs or cats and cause serious human infections. We aimed to evaluate the ability of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to identify these two Capnocytophaga species. Ninety-four C. canimorsus and 10 C. cynodegmi isolates identified by 16S rRNA gene sequencing were analyzed. Using the MALDI BioTyper database, correct identification was achieved for only 16 of 94 (17%) C. canimorsus and all 10 C. cynodegmi strains, according to the manufacturer's log score specifications. Following the establishment of a complementary homemade reference database by addition of 51 C. canimorsus and 8 C. cynodegmi mass spectra, MALDI-TOF MS provided reliable identification to the species level for 100% of the 45 blind-coded Capnocytophaga isolates tested. MALDI-TOF MS can accurately identify C. canimorsus and C. cynodegmi using an enriched database and thus constitutes a valuable diagnostic tool in the clinical laboratory.

Modification of the 1-Phosphate Group during Biosynthesis of Capnocytophaga canimorsus Lipid A.

Capnocytophaga canimorsus, a commensal bacterium of dog's mouth flora causing severe infections in humans after dog bites or scratches, has a lipopolysaccharide (LPS) (endotoxin) with low-inflammatory lipid A. In particular, it contains a phosphoethanolamine (P-Etn) instead of a free phosphate group at the C-1 position of the lipid A backbone, usually present in highly toxic enterobacterial Gram-negative lipid A. Here we show that the C. canimorsus genome comprises a single operon encoding a lipid A 1-phosphatase (LpxE) and a lipid A 1 P-Etn transferase (EptA). This suggests that lipid A is modified during biosynthesis after completing acylation of the backbone by removal of the 1-phosphate and subsequent addition of an P-Etn group. As endotoxicity of lipid A is known to depend largely on the degree of unsubstituted or unmodified phosphate residues, deletion of lpxE or eptA led to mutants lacking the P-Etn group, with consequently increased endotoxicity and decreased resistance to cationic antimicrobial peptides (CAMP). Consistent with the proposed sequential biosynthetic mechanism, the endotoxicity and CAMP resistance of a double deletion mutant of lpxE-eptA was similar to that of a single lpxE mutant. Finally, the proposed enzymatic activities of LpxE and EptA based on sequence similarity could be successfully validated by mass spectrometry (MS)-based analysis of lipid A isolated from the corresponding deletion mutant strains.

Only a subset of C. canimorsus strains is dangerous for humans.

Capnocytophaga canimorsus are gram-negative bacteria living as commensals in the mouth of dogs and cats. C. canimorsus cause rare but life-threatening generalized infections in humans that have been in contact with a dog or a cat. Over the last years we collected 105 C. canimorsus strains from different geographical origins and from severe human infections or healthy dogs. All these strains were analyzed by 16S rDNA sequencing and a phylogenetic tree revealed two main groups of bacteria instead of one with no relation to the geographical origin. This branching was confirmed by the whole-genome sequencing of 10 strains, supporting the evidence of a new Capnocytophaga species in dogs. Interestingly, 19 out of 19 C. canimorsus strains isolated from human infections belonged to the same species. Furthermore, most strains from this species could grow in heat-inactivated human serum (HIHS) (40/46 tested), deglycosylate IgM (48/66) and were cytochrome-oxidase positive (60/66) while most strains from the other species could not grow in HIHS (22/23 tested), could not deglycosylate IgM (33/34) and were cytochrome-oxidase negative (33/34). Here, we propose to call Capnocytophaga canis (Latin: dog) the novel, presumably less virulent dog-hosted Capnocytophaga species and to keep the name C. canimorsus for the species including human pathogens.

Draft Genome Sequences of Three Capnocytophaga canimorsus Strains Isolated from Septic Patients.

Capnocytophaga canimorsus is a bacterium from the normal oral flora of dogs and cats that causes rare generalized infections in humans. In an attempt to determine whether infections could be caused by a subset of strains and to identify pathogenicity factors, we sequenced the genomes of three strains isolated from human infections.