Facial Soft Tissue Lesions in Children.

Facial soft tissue lesions in children are often classified based on their structure or cellular origin and can be benign or malignant. This review focuses on common facial soft tissue lesions in children, their clinical morphology, natural history, and medical and surgical management, with an emphasis on those considerations unique to soft tissue lesions present at this anatomic site.

Second Harmonic Generation Microscopy as a Novel Intraoperative Assessment of Rat Median Nerve Injury.

PURPOSE: Nerves that are functionally injured but appear macroscopically intact pose the biggest clinical dilemma. Second Harmonic Generation (SHG) Microscopy may provide a real-time assessment of nerve damage, with the ultimate goal of allowing surgeons to accurately quantify the degree of nerve damage present. The aim of this study was to demonstrate the utility of SHG microscopy to detect nerve damage in vivo in an animal model. METHODS: Ten Sprague-Dawley rats were anesthetized and prepared for surgery. After surgical exposure and using a custom-made stretch applicator, the right median nerves were stretched by 20%, corresponding to a high strain injury, and held for 5 minutes. The left median nerve served as a sham control (SC), only being placed in the applicator for 5 minutes with no stretch. A nerve stimulator was used to assess the amount of stimulation required to induce a flicker and contraction of the paw. Nerves were then imaged using a multiphoton laser scanning microscope. RESULTS: Immediately after injury (day 0), SHG images of SC median nerves exhibited parallel collagen fibers with linear, organized alignment. In comparison with SC nerves, high strain nerves demonstrated artifacts indicative of nerve damage consisting of wavy, undulating fibers with crossing fibers and tears, as well as a decrease in the linear organization, which correlated with an increase in the mean stimulation required to induce a flicker and contraction of the paw. CONCLUSIONS: Second Harmonic Generation microscopy may provide the ability to detect an acute neural stretch injury in the rat median nerve. Epineurial collagen disorganization correlated with the stimulation required for nerve function. CLINICAL RELEVANCE: In the future, SHG may provide the ability to visualize nerve damage intraoperatively, allowing for better clinical decision-making. However, this is currently a research tool and requires further validation before translating to the clinical setting.

Outcomes of Arthroscopic Elbow Contracture Release: Improvement for Severe Prosupination and Flexion Contracture.

PURPOSE: The purpose of this study was to investigate outcomes following arthroscopic elbow contracture release to describe the use of arthroscopy for improvement in extension/flexion and pronation/supination arcs of motion at a single institution for degenerative and posttraumatic etiologies. METHODS: Consecutive arthroscopic elbow arthrolysis performed between 2003 and 2015 were retrospectively reviewed. Basic patient demographics, indications for surgery, preoperative and postoperative elbow range of motion, postoperative patient outcome score, and all complications were recorded and analyzed. RESULTS: Fifty-two patients were included with an average follow-up of 5.1 years (range 1.4 to 9.4). Severe contractures made up 50% of cases, followed by 23% moderate, and 27% mild. Average extension/flexion for the post-traumatic group (n = 30) increased by 63° ± 31 and by 29° ± 24 for the degenerative group (n = 22). Average gain in pronosupination was 38° ± 62 in the post-traumatic group and 13°±23 in the degenerative group. Postoperative DASH scores were 17.5 ± 18.4 for post-traumatic cases and 12.8 ± 19.3 for degenerative cases. CONCLUSION: Arthroscopic elbow contracture release is an effective intervention for degenerative and post-traumatic elbow contracture for both flexion/extension and pronosupination contracture. Furthermore, a two-stage release should be considered when both flexion and pronosupinaton contractures are present. LEVEL OF EVIDENCE: IV, case series, treatment study.

The ß-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion.

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large ß-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs- mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro-Ala-Ala-Arg (PAAR) repeat domains, which bind the ß-spike of trimeric valine-glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact ß-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs+ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the ß-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact ß-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

The Early Effects of COVID-19 on Plastic Surgery Residency Training: The University of Washington Experience.

Plastic surgeons have the unique perspective of working with all types of patients and care teams from almost all specialties in surgery and medicine, which creates unique challenges in times of distress. As the initial epicenter of coronavirus disease 2019 cases in the United States, the University of Washington program was required to rapidly develop strategies to deal with the escalating crisis. All aspects of the program were affected, including the need to triage the urgency of plastic surgery care, safe staffing of plastic surgery teams, and the role of plastic surgery in the greater hospital community. In addition, as a residency training program, limiting the impact of resident education and maintaining a sense of community and connection among members of the program developed into important considerations. The authors hope that the narrative of their experience will provide insight into the decisions made in the University of Washington health care system but also remind others that they are not alone in dealing with the challenges of this pandemic.

Intramedullary Screw Fixation of Metacarpal Fractures Results in Excellent Functional Outcomes: A Literature Review.

BACKGROUND: Intramedullary fixation of a comminuted subcapital metacarpal fracture using a headless compression screw was first described in 2010. The purpose of this study was to identify and evaluate all studies reporting clinical outcomes of intramedullary screw fixation of metacarpal fractures. METHODS: A comprehensive search of the literature was performed under Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines to identify all relevant studies. Outcome measures included mean follow-up, evidence of radiographic union, functional outcomes, and quality-of-life measures. RESULTS: Nine articles for a total of 169 metacarpal fractures were identified. Average patient age was 32 years; 86.5 percent of the patients were men; and the majority of fractures occurred in the small finger (74 percent). Most fractures occurred in the neck (n = 66), followed by shaft (n = 31) and head (n = 10). The average follow-up was 11 months, with an average metacarpophalangeal joint flexion of 86 degrees (n = 83) and digit total active motion of 251 degrees (n = 72). Radiographic union was achieved in 100% of reported cases (n = 132) at or before the latest follow-up. Grip strength in four studies showed an average of 96 percent compared to the contralateral hand. No serious complications were reported. Nine minor complications were reported, including four cases of hardware removal in asymptomatic patients. CONCLUSION: The review of the literature suggests intramedullary fixation of metacarpal neck and shaft fractures using headless compression screws has thus far proven to be a safe and successful surgical treatment option resulting in excellent clinical outcomes.

Comparative strength of elbow splint designs: a new splint design as a stronger alternative to posterior splints.

BACKGROUND: Musculoskeletal injuries of the upper extremity are frequently treated with temporary external immobilization. Traditionally, long arm posterior splints have been used to limit flexion/extension of the elbow. However, long arm posterior splints have been observed to fail clinically, necessitating a stronger alternative. In this study, we assessed the biomechanical strength of the long arm posterior splint compared with a new spiral splint design. METHODS: One male and one female participant were placed 10 times in long arm posterior splints and 10 times in spiral splints. Both splint types were subjected to a downward mechanical load of 39.2 N (4 kg) and assessed for a change in both flexion/extension and pronation/supination. RESULTS: There was no significant difference in starting position or starting flexion/extension between the 2 splint designs. Posterior splints allowed significantly greater initial pronation/supination compared with spiral splints. Both splint groups had significant increases in flexion/extension and pronation/supination compared with their starting ranges of motion. There was no significant difference in the change in pronation/supination between the 2 splint groups. Finally, posterior splints allowed a significantly greater change in flexion/extension compared with spiral splints. CONCLUSION: Spiral splints offered less initial pronation/supination than long arm posterior splints. Furthermore, spiral splints are able to resist flexion/extension of the elbow after application of a downward mechanical load better than posterior splints, thus suggesting spiral splints are mechanically superior to long arm posterior splints.

Varus posteromedial rotatory instability: a biomechanical analysis of posterior bundle of the medial ulnar collateral ligament reconstruction.

BACKGROUND: Recently, there has been growing interest in the involvement of the posterior bundle of the medial ulnar collateral ligament (pMUCL) in varus posteromedial rotatory instability (PMRI). Varus PMRI has been observed clinically, but the degree of involvement of the pMUCL remains unclear. This study assessed the degree to which the pMUCL is involved in stabilizing the elbow and the feasibility of a pMUCL reconstruction to restore stability. METHODS: Movements simulating PMRI were performed in 8 cadaveric elbows. Joint gapping values were obtained by 3-dimensional motion capture for the proximal and distal aspects of the ulnohumeral joint. Specimens were assessed at "intact," "cut coronoid + pMUCL," "reconstruction," and "cut anterior aspect MUCL + reconstruction" conditions with mechanical testing at 30°, 60°, and 90° of elbow flexion. RESULTS: Proximal joint gapping significantly increased from intact to cut coronoid + pMUCL at 60° and 90°, and distal joint gapping significantly increased at 90°. In the reconstruction condition, joint gapping across the proximal joint at 60° and 90° significantly recovered, as did distal joint gapping at 90°. In the cut anterior aspect MUCL + reconstruction condition, no significant increase occurred in proximal or distal joint gapping. CONCLUSIONS: Transection of the pMUCL with a coronoid fracture leads to increased joint gapping, suggesting the presence of PMRI. PMRI can still occur with an intact lateral ligamentous complex. A pMUCL tendon graft reconstruction confers some elbow stability in this injury mechanism.

The effect of screw trajectory for the reduction and association of the scaphoid and lunate (RASL) procedure: a biomechanical analysis.

The purpose of this study was to determine if screw placement in the reduction and association of the scaphoid and the lunate (RASL) procedure affected the ability of the scapholunate joint to withstand force. After completely disrupting of the scapholunate ligament in 29 fresh-frozen cadaveric wrists, we placed the RASL screw either distal or proximal to the lateral aspect of the dorsal ridge of the scaphoid and into the dorsal or volar aspect of the lunate. Specimens were subjected to repeated cycles of transcarpal axial force, mimicking clenched-fist loading, until failure. Screw placement distal to the lateral aspect of the dorsal scaphoid ridge was significantly associated with failure when examined manually, radiographically (1.8 vs. 0.5 mm) and using real-time motion capture (diastasis: 1.6 vs. 0.4 mm; Euler angle: 4.5 ° vs. 0.8 °). The lateral aspect of the dorsal ridge is a reliable radiographic landmark on the scaphoid and provides surgeons with a convenient starting point to achieve the most biomechanically stable RASL construct, and, therefore, enhances the potential for an optimal clinical outcome.

Activation of an anti-bacterial toxin by the biosynthetic enzyme CysK: mechanism of binding, interaction specificity and competition with cysteine synthase.

Contact-dependent growth inhibition (CDI) is a wide-spread mechanism of inter-bacterial competition. CDI+ bacteria deliver CdiA-CT toxins into neighboring bacteria and produce specific immunity proteins that protect against self-intoxication. The CdiA-CT toxin from uropathogenic Escherichia coli 536 is a latent tRNase that is only active when bound to the cysteine biosynthetic enzyme CysK. Remarkably, the CysK:CdiA-CT binding interaction mimics the 'cysteine synthase' complex of CysK:CysE. The C-terminal tails of CysE and CdiA-CT each insert into the CysK active-site cleft to anchor the respective complexes. The dissociation constant for CysK:CdiA-CT (K d ~ 11 nM) is comparable to that of the E. coli cysteine synthase complex (K d ~ 6 nM), and both complexes bind through a two-step mechanism with a slow isomerization phase after the initial encounter. However, the second-order rate constant for CysK:CdiA-CT binding is two orders of magnitude slower than that of the cysteine synthase complex, suggesting that CysE should outcompete the toxin for CysK occupancy. However, we find that CdiA-CT can effectively displace CysE from pre-formed cysteine synthase complexes, enabling toxin activation even in the presence of excess competing CysE. This adventitious binding, coupled with the very slow rate of CysK:CdiA-CT dissociation, ensures robust nuclease activity in target bacteria.

CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria.

Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiAEC93 from Escherichia coli EC93 and OmpC-specific CdiAEC536 from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiAEC93 fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiAEC93 that carries the corresponding sequence from CdiAEC536 is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads.IMPORTANCE CdiB/CdiA two-partner secretion proteins mediate interbacterial competition through the delivery of polymorphic toxin domains. This process, known as contact-dependent growth inhibition (CDI), requires stable interactions between the CdiA effector protein and specific receptors on the surface of target bacteria. Here, we localize the receptor-binding domain to the central region of E. coli CdiA. Receptor-binding domains vary between CdiA proteins, and E. coli strains collectively encode at least four distinct effector classes. Further, we show that receptor specificity can be altered by exchanging receptor-binding regions, demonstrating the modularity of this domain. We propose that novel CdiA effectors are naturally generated through genetic recombination to interchange different receptor-binding domains and toxin payloads.

CdiA Effectors from Uropathogenic Escherichia coli Use Heterotrimeric Osmoporins as Receptors to Recognize Target Bacteria.

Many Gram-negative bacterial pathogens express contact-dependent growth inhibition (CDI) systems that promote cell-cell interaction. CDI+ bacteria express surface CdiA effector proteins, which transfer their C-terminal toxin domains into susceptible target cells upon binding to specific receptors. CDI+ cells also produce immunity proteins that neutralize the toxin domains delivered from neighboring siblings. Here, we show that CdiAEC536 from uropathogenic Escherichia coli 536 (EC536) uses OmpC and OmpF as receptors to recognize target bacteria. E. coli mutants lacking either ompF or ompC are resistant to CDIEC536-mediated growth inhibition, and both porins are required for target-cell adhesion to inhibitors that express CdiAEC536. Experiments with single-chain OmpF fusions indicate that the CdiAEC536 receptor is heterotrimeric OmpC-OmpF. Because the OmpC and OmpF porins are under selective pressure from bacteriophages and host immune systems, their surface-exposed loops vary between E. coli isolates. OmpC polymorphism has a significant impact on CDIEC536 mediated competition, with many E. coli isolates expressing alleles that are not recognized by CdiAEC536. Analyses of recombinant OmpC chimeras suggest that extracellular loops L4 and L5 are important recognition epitopes for CdiAEC536. Loops L4 and L5 also account for much of the sequence variability between E. coli OmpC proteins, raising the possibility that CDI contributes to the selective pressure driving OmpC diversification. We find that the most efficient CdiAEC536 receptors are encoded by isolates that carry the same cdi gene cluster as E. coli 536. Thus, it appears that CdiA effectors often bind preferentially to "self" receptors, thereby promoting interactions between sibling cells. As a consequence, these effector proteins cannot recognize nor suppress the growth of many potential competitors. These findings suggest that self-recognition and kin selection are important functions of CDI.

Unraveling the essential role of CysK in CDI toxin activation.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of bacterial competition. CDI(+) bacteria deliver the toxic C-terminal region of contact-dependent inhibition A proteins (CdiA-CT) into neighboring target bacteria and produce CDI immunity proteins (CdiI) to protect against self-inhibition. The CdiA-CT(EC536) deployed by uropathogenic Escherichia coli 536 (EC536) is a bacterial toxin 28 (Ntox28) domain that only exhibits ribonuclease activity when bound to the cysteine biosynthetic enzyme O-acetylserine sulfhydrylase A (CysK). Here, we present crystal structures of the CysK/CdiA-CT(EC536) binary complex and the neutralized ternary complex of CysK/CdiA-CT/CdiI(EC536) CdiA-CT(EC536) inserts its C-terminal Gly-Tyr-Gly-Ile peptide tail into the active-site cleft of CysK to anchor the interaction. Remarkably, E. coli serine O-acetyltransferase uses a similar Gly-Asp-Gly-Ile motif to form the "cysteine synthase" complex with CysK. The cysteine synthase complex is found throughout bacteria, protozoa, and plants, indicating that CdiA-CT(EC536) exploits a highly conserved protein-protein interaction to promote its toxicity. CysK significantly increases CdiA-CT(EC536) thermostability and is required for toxin interaction with tRNA substrates. These observations suggest that CysK stabilizes the toxin fold, thereby organizing the nuclease active site for substrate recognition and catalysis. By contrast, Ntox28 domains from Gram-positive bacteria lack C-terminal Gly-Tyr-Gly-Ile motifs, suggesting that they do not interact with CysK. We show that the Ntox28 domain from Ruminococcus lactaris is significantly more thermostable than CdiA-CT(EC536), and its intrinsic tRNA-binding properties support CysK-independent nuclease activity. The striking differences between related Ntox28 domains suggest that CDI toxins may be under evolutionary pressure to maintain low global stability.

Moonlighting O-acetylserine sulfhydrylase: New functions for an old protein.

O-acetylserine sulfhydrylase A (CysK) is the pyridoxal 5'-phosphate-dependent enzyme that catalyzes the final reaction of cysteine biosynthesis in bacteria. CysK was initially identified in a complex with serine acetyltransferase (CysE), which catalyzes the penultimate reaction in the synthetic pathway. This "cysteine synthase" complex is stabilized by insertion of the CysE C-terminus into the active-site of CysK. Remarkably, the CysK/CysE binding interaction is conserved in most bacterial and plant systems. For the past 40years, CysK was thought to function exclusively in cysteine biosynthesis, but recent studies have revealed a repertoire of additional "moonlighting" activities for this enzyme. CysK and its paralogs influence transcription in both Gram-positive bacteria and the nematode Caenorhabditis elegans. CysK also activates an antibacterial nuclease toxin produced by uropathogenic Escherichia coli. Intriguingly, each moonlighting activity requires a binding partner that invariably mimics the C-terminus of CysE to interact with the CysK active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

The proton-motive force is required for translocation of CDI toxins across the inner membrane of target bacteria.

Contact-dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two-partner secretion proteins. The CdiA effector extends from the surface of CDI(+) inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C-terminal region (CdiA-CT). Here, we show that CdiA-CT toxin translocation requires the proton-motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA-CT translocation is clearly distinct from known colicin-import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA-CT toxins can be transferred into the periplasm of de-energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de-energized conditions remain competent to enter the target-cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer- and inner-membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target-cell cytoplasm.

The F pilus mediates a novel pathway of CDI toxin import.

Contact-dependent growth inhibition (CDI) is a widespread form of inter-bacterial competition that requires direct cell-to-cell contact. CDI(+) inhibitor cells express CdiA effector proteins on their surface. CdiA binds to specific receptors on susceptible target bacteria and delivers a toxin derived from its C-terminal region (CdiA-CT). Here, we show that purified CdiA-CT(536) toxin from uropathogenic Escherichia coli 536 translocates into bacteria, thereby by-passing the requirement for cell-to-cell contact during toxin delivery. Genetic analyses demonstrate that the N-terminal domain of CdiA-CT(536) is necessary and sufficient for toxin import. The CdiA receptor plays no role in this import pathway; nor do the Tol and Ton systems, which are exploited to internalize colicin toxins. Instead, CdiA-CT(536) import requires conjugative F pili. We provide evidence that the N-terminal domain of CdiA-CT(536) interacts with F pilin, and that pilus retraction is critical for toxin import. This pathway is reminiscent of the strategy used by small RNA leviviruses to infect F(+) cells. We propose that CdiA-CT(536) mimics the pilin-binding maturation proteins of leviviruses, allowing the toxin to bind F pili and become internalized during pilus retraction.

Genetically distinct pathways guide effector export through the type VI secretion system.

Bacterial secretion systems often employ molecular chaperones to recognize and facilitate export of their substrates. Recent work demonstrated that a secreted component of the type VI secretion system (T6SS), haemolysin co-regulated protein (Hcp), binds directly to effectors, enhancing their stability in the bacterial cytoplasm. Herein, we describe a quantitative cellular proteomics screen for T6S substrates that exploits this chaperone-like quality of Hcp. Application of this approach to the Hcp secretion island I-encoded T6SS (H1-T6SS) of Pseudomonas aeruginosa led to the identification of a novel effector protein, termed Tse4 (type VI secretion exported 4), subsequently shown to act as a potent intra-specific H1-T6SS-delivered antibacterial toxin. Interestingly, our screen failed to identify two predicted H1-T6SS effectors, Tse5 and Tse6, which differ from Hcp-stabilized substrates by the presence of toxin-associated PAAR-repeat motifs and genetic linkage to members of the valine-glycine repeat protein G (vgrG) genes. Genetic studies further distinguished these two groups of effectors: Hcp-stabilized effectors were found to display redundancy in interbacterial competition with respect to the requirement for the two H1-T6SS-exported VgrG proteins, whereas Tse5 and Tse6 delivery strictly required a cognate VgrG. Together, we propose that interaction with either VgrG or Hcp defines distinct pathways for T6S effector export.

CdiA from Enterobacter cloacae delivers a toxic ribosomal RNase into target bacteria.

Contact-dependent growth inhibition (CDI) is one mechanism of inter-bacterial competition. CDI(+) cells export large CdiA effector proteins, which carry a variety of C-terminal toxin domains (CdiA-CT). CdiA-CT toxins are specifically neutralized by cognate CdiI immunity proteins to protect toxin-producing cells from autoinhibition. Here, we use structure determination to elucidate the activity of a CDI toxin from Enterobacter cloacae (ECL). The structure of CdiA-CT(ECL) resembles the C-terminal nuclease domain of colicin E3, which cleaves 16S ribosomal RNA to disrupt protein synthesis. In accord with this structural homology, we show that CdiA-CT(ECL) uses the same nuclease activity to inhibit bacterial growth. Surprisingly, although colicin E3 and CdiA(ECL) carry equivalent toxin domains, the corresponding immunity proteins are unrelated in sequence, structure, and toxin-binding site. Together, these findings reveal unexpected diversity among 16S rRNases and suggest that these nucleases are robust and versatile payloads for a variety of toxin-delivery platforms.

Microdroplet sandwich real-time rt-PCR for detection of pandemic and seasonal influenza subtypes.

As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR). Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic), H1N1s (seasonal), and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 10(4) copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies.

Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI).

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI(+) inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI(+) cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK(+) cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.