Dual-Toxin ("Bivalent") Infant Botulism in California, 1976-2020: Epidemiologic, Clinical, and Laboratory Aspects.

OBJECTIVE: To assess the consequences of infant botulism that result from Clostridium botulinum strains that produce 2 botulinum toxin serotypes, termed "bivalent." STUDY DESIGN: Epidemiologic investigations used a standard questionnaire. Clostridium botulinum strains were isolated by standard methods. Botulinum neurotoxin (BoNT) serotypes and the relative amounts of toxins produced were identified using the standard mouse bioassay. BoNT subtypes and genomic locations were identified by DNA nucleotide sequencing. RESULTS: Thirty bivalent cases of infant botulism occurred in the 45 years (1976-2020), representing 2.0% of all California infant botulism cases, in the 3 geographic regions of southern California, the southern Central Valley, and mid-northern California. Toxin serotype combinations were Ba (n = 22), Bf (n = 7), and Ab (n = 1). More patients with illness caused by bivalent C botulinum Ba and Bf strains needed endotracheal intubation at hospital admission, 60.0% (18/30), than did patients with illness caused by monovalent BoNT/B strains, 34.3% (152/443). The Cbotulinum Ba and Bf strains produced BoNT/B5 and either BoNT/A4 or /F2. The Ab strain produced BoNT/A2 and /B1. All toxin gene clusters were on plasmids. CONCLUSIONS: Infant botulism caused by bivalent Cbotulinum strains occurs sporadically and in diverse locations in California. Affected patients with bivalent Ba and Bf strains lacked distinguishing epidemiological features but appeared to be more severely paralyzed at hospital presentation than patients with illness caused by only BoNT/B. These bivalent strains produced BoNT subtypes A2, A4, B1, B5, and F2, and all toxin gene clusters were on plasmids.

The Role of Protein Loss and Denaturation in Determining Outcomes of Heating, Cryotherapy, and Irreversible Electroporation on Cardiomyocytes.

Atrial fibrillation (AF) currently affects millions of people in the U.S. alone. Focal therapy is an increasingly attractive treatment for AF that avoids the debilitating effects of drugs for disease control. Perhaps the most widely used focal therapy for AF is heat-based radiofrequency (heating), although cryotherapy (cryo) is rapidly replacing it due to a reduction in side effects and positive clinical outcomes. A third focal therapy, irreversible electroporation (IRE), is also being considered in some settings. This study was designed to help guide treatment thresholds and compare mechanism of action across heating, cryo, and IRE. Testing was undertaken on HL-1 cells, a well-established cardiomyocyte cell line, to assess injury thresholds for each treatment method. Cell viability, as assessed by Hoechst and propidium iodide (PI) staining, was found to be minimal after exposure to temperatures ≤-40 °C (cryo), ≥60 °C (heating), and when field strengths ≥1500 V/cm (IRE) were used. Viability was then correlated to protein denaturation fraction (PDF) as assessed by Fourier transform infrared (FTIR) spectroscopy, and protein loss fraction (PLF) as assessed by bicinchoninic acid (BCA) assay after the three treatments. These protein changes were assessed both in the supernatant and the pellet of cell suspensions post-treatment. We found that dramatic viability loss (≥50%) correlated strongly with ≥12% protein change (PLF, PDF or a combination of the two) in every focal treatment. These studies help in defining both cellular thresholds and protein-based mechanisms of action that can be used to improve focal therapy application for AF.

Identification of Novel 14-3-3 Residues That Are Critical for Isoform-specific Interaction with GluN2C to Regulate N-Methyl-D-aspartate (NMDA) Receptor Trafficking.

The 14-3-3 family of proteins is widely distributed in the CNS where they are major regulators of essential neuronal functions. There are seven known mammalian 14-3-3 isoforms (ζ,, τ, ϵ, η, ß, and σ), which generally function as adaptor proteins. Previously, we have demonstrated that 14-3-3ϵ isoform dynamically regulates forward trafficking of GluN2C-containing NMDA receptors (NMDARs) in cerebellar granule neurons, that when expressed on the surface, promotes neuronal survival following NMDA-induced excitotoxicity. Here, we report 14-3-3 isoform-specific binding and functional regulation of GluN2C. In particular, we show that GluN2C C-terminal domain (CTD) binds to all 14-3-3 isoforms except 14-3-3σ, and binding is dependent on GluN2C serine 1096 phosphorylation. Co-expression of 14-3-3 (ζ and ϵ) and GluN1/GluN2C promotes the forward delivery of receptors to the cell surface. We further identify novel residues serine 145, tyrosine 178, and cysteine 189 on α-helices 6, 7, and 8, respectively, within ζ-isoform as part of the GluN2C binding motif and independent of the canonical peptide binding groove. Mutation of these conserved residues abolishes GluN2C binding and has no functional effect on GluN2C trafficking. Reciprocal mutation of alanine 145, histidine 180, and isoleucine 191 on 14-3-3σ isoform promotes GluN2C binding and surface expression. Moreover, inhibiting endogenous 14-3-3 using a high-affinity peptide inhibitor, difopein, greatly diminishes GluN2C surface expression. Together, these findings highlight the isoform-specific structural and functional differences within the 14-3-3 family of proteins, which determine GluN2C binding and its essential role in targeting the receptor to the cell surface to facilitate glutamatergic neurotransmission.

ERF and ETV3L are retinoic acid-inducible repressors required for primary neurogenesis.

Cells in the developing neural tissue demonstrate an exquisite balance between proliferation and differentiation. Retinoic acid (RA) is required for neuronal differentiation by promoting expression of proneural and neurogenic genes. We show that RA acts early in the neurogenic pathway by inhibiting expression of neural progenitor markers Geminin and Foxd4l1, thereby promoting differentiation. Our screen for RA target genes in early Xenopus development identified Ets2 Repressor Factor (Erf) and the closely related ETS repressors Etv3 and Etv3-like (Etv3l). Erf and Etv3l are RA responsive and inhibit the action of ETS genes downstream of FGF signaling, placing them at the intersection of RA and growth factor signaling. We hypothesized that RA regulates primary neurogenesis by inducing Erf and Etv3l to antagonize proliferative signals. Loss-of-function analysis showed that Erf and Etv3l are required to inhibit proliferation of neural progenitors to allow differentiation, whereas overexpression of Erf led to an increase in the number of primary neurons. Therefore, these RA-induced ETS repressors are key components of the proliferation-differentiation switch during primary neurogenesis in vivo.

Predictable Heating and Positive MRI Contrast from a Mesoporous Silica-Coated Iron Oxide Nanoparticle.

Iron oxide nanoparticles have great potential as diagnostic and therapeutic agents in cancer and other diseases; however, biological aggregation severely limits their function in vivo. Aggregates can cause poor biodistribution, reduced heating capability, and can confound their visualization and quantification by magnetic resonance imaging (MRI). Herein, we demonstrate that the incorporation of a functionalized mesoporous silica shell can prevent aggregation and enable the practical use of high-heating, high-contrast iron oxide nanoparticles in vitro and in vivo. Unmodified and mesoporous silica-coated iron oxide nanoparticles were characterized in biologically relevant environments including phosphate buffered saline, simulated body fluid, whole mouse blood, lymph node carcinoma of prostate (LNCaP) cells, and after direct injection into LNCaP prostate cancer tumors in nude mice. Once coated, iron oxide nanoparticles maintained colloidal stability along with high heating and relaxivity behaviors (SARFe = 204 W/g Fe at 190 kHz and 20 kA/m and r1 = 6.9 mM(-1) s(-1) at 1.4 T). Colloidal stability and minimal nonspecific cell uptake allowed for effective heating in salt and agarose suspensions and strong signal enhancement in MR imaging in vivo. These results show that (1) aggregation can lower the heating and imaging performance of magnetic nanoparticles and (2) a coating of functionalized mesoporous silica can mitigate this issue, potentially improving clinical planning and practical use.

A strategy for the successful management of dermatofibrosarcoma protuberans.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a rare, locally invasive soft tissue sarcoma with extensive subclinical involvement. The National Comprehensive Cancer Network guidelines recommend immediate reconstruction in most cases. Our study reviewed the methods of treatment of DFSP at our institution, examined the types of closure used after surgical excision, and analyzed the prevalence of positive margins on permanent pathology after immediate closure after conventional non-Mohs excision of DFSP. METHODS: The charts of 25 patients treated with surgical excision and 16 with Mohs surgery from 1990 to 2009 for lesions consistent with DFSP were reviewed for clinical variables including disease state, tumor site, closure type, permanent pathology margin status, disease recurrence/persistence, and excisional margin size. RESULTS: The trunk, followed by the head and neck, were the most common sites for DFSP. No patients had distant metastasis at diagnosis or experienced recurrence in either the surgical excision or the Mohs surgery group. Twelve (48%) patients were found to have positive margins after initial surgical resection. All lesions treated with Mohs surgery had clear histological margins at completion. Average margin size for surgical excision patients was 2.33 cm (range, 0.75-4.5 cm), and 1.36 cm (range, 0.74-2.55 cm) for Mohs excision. The average duration of follow-up was 107.9 months. CONCLUSIONS: The extent of DFSP is difficult to determine intraoperatively with traditional surgical excision, which leads to a higher rate of positive margins. Considering this difficulty and the complications of reconstruction with positive margins, we believe that reconstruction after tumor resection should be dependent on definitive pathologic clearance of the tumor.

A randomized, double-blind, placebo-controlled, pilot study to assess the efficacy and safety of clindamycin 1.2% and tretinoin 0.025% combination gel for the treatment of acne rosacea over 12 weeks.

BACKGROUND: Papulopustular acne rosacea is a chronic inflammatory condition which can be difficult to treat. Many patients are unwilling to use systemic medications, and single topical agents alone may not address all the symptoms of rosacea. A combination topical clindamycin phosphate 1.2% and tretinoin 0.025% gel is efficacious for acne vulgaris, and may be helpful for rosacea, since acne vulgaris and rosacea shares many similar clinical and histologic features. OBJECTIVE: To assess the preliminary efficacy and safety of a combination gel consisting of clindamycin phosphate 1.2% and tretinoin 0.025% on papulopustular rosacea after 12 weeks of usage. METHODS: Randomized, double-blind, placebo controlled two site study of 79 participants with moderate to severe papulopustular acne rosacea using both physician and subjects' validated assessment tools. Primary endpoint consisted of statistically significant reduction in absolute papule or pustule count after 12 weeks of usage. RESULTS: There was no significant difference in papule/pustule count between placebo and treated groups after 12 weeks (P=0.10). However, there was nearly significant improvement in physicians' assessments of the telangiectasia component of rosacea (P=0.06) and erythematotelangiectatic rosacea subtype (P=0.05) in treated versus placebo group after 12 weeks. The only significant adverse event different was facial scaling, which was significantly increased in treated group (P=0.01), but this did not result in discontinuation of study drug. CONCLUSIONS: A combination gel of clindamycin phosphate 1.2% and tretinoin 0.025% may improve the telangiectatic component of rosacea and appears to better treat the erythemotelangiectatic subtype of rosacea rather than papulopustular subtype. Our preliminary study suggests that future studies with much larger sample size might confirm our findings.

Human amniotic membrane modulates Wnt/ß-catenin and NF-κß signaling pathways in articular chondrocytes in vitro.

Objective: Inflammation, catabolism, and hypertrophy in chondrocytes play a central role in osteoarthritis (OA). The Wnt/ß-catenin and NF-κß pathways contribute to these degradative processes. This in vitro study evaluates the inhibitory effect of a novel therapeutic, micronized dehydrated human amnion/chorion membrane (µdHACM), as a potential treatment to offset elevated Wnt/ß-catenin and NF-κß signaling. Design: Three-dimensional human articular chondrocyte pellets were stimulated with an inflammatory cocktail to induce a degenerative phenotype. Treatments included varying doses of µdHACM. Protein and gene expression were analyzed using qRT-PCR, immunoblotting, and immunofluorescence to assess changes in the major constituents of Wnt/ß-catenin and NF-κß signaling. Regulation of catabolic activity was evaluated using enzymatic assays that detect MMP-13 and aggrecanase-mediated degradation products in conditioned media. Results: Confirmation of the model was established through the expression of specific markers and extracellular matrix genes, verifying a chondrogenic phenotype was maintained. Inflammatory stimulation elicited a change in the chondrocyte proteome and secretome, elevating Wnt/ß-catenin and NF-κß signaling and downstream expression of inflammatory, proteolytic, and hypertrophic markers, while decreasing primary cartilage matrix components, ACAN and COL2A1. µdHACM reversed these inflammatory-induced changes, suppressing phospho-GSK-3ß, ß-catenin expression/nuclear localization of the Wnt signaling axis and inhibiting IKKß, phospho-IκBα, and phospho-p65 in the NF-κß signaling cascade. Additionally, µdHACM altered expression of direct downstream targets, namely MCP1, MMP3, MMP13, ADAMTS4, ADAMTS5, RUNX2 and COL10A1. Moreover, µdHACM reduced MMP-13 and aggrecanase-mediated substrate degradation. Conclusion: µdHACM ameloriated the effects of inflammatory-induced degeneration in chondrocytes through Wnt/ß-catenin and NF-κß inhibition, subsequently downregulating key inflammatory, hypertrophic and catabolic mediators in vitro.

A Case of Posterior Nutcracker Syndrome Revealed in the Aerospace Environment.

BACKGROUND: Nutcracker syndrome is caused by a rare anatomic variant where the left renal vein is trapped between the aorta and the superior mesenteric artery. Posterior nutcracker syndrome is an even rarer entity, characterized by the retro-aortic positioning of the renal vein, causing compression between the aorta and spinal vertebrae. Symptoms include microscopic or frank hematuria, flank pain, varicocele, pelvic congestion syndrome, and abdominal pain. A search of the literature did not reveal prior cases of nutcracker syndrome that became symptomatic and diagnosed secondary to the unique stressors of high gravitational force (G force) in the aviation environment.CASE REPORT: A 25-yr-old man training as an F-16 flight test engineer presented with left scrotal/testicular pain, varicocele, and intermittent gross hematuria. After an extensive workup, he was diagnosed with posterior nutcracker syndrome and underwent a left varicocele ligation with spermatic cord denervation. He was eventually able to be returned to flying duties with limitation to non-high performance aircraft.DISCUSSION: This case is particularly unique as its diagnosis was dependent on exposure to high G force conditions that may have otherwise remained asymptomatic without this environmental stressor. Education on the diagnosis of nutcracker syndrome as a differential in the setting of hematuria and pain is an important lesson learned. This case also illustrates the necessity of considering the effects of the stressful environment of high G force on even overall healthy individuals. Fortunately, due to the collaboration of medical-surgical expertise and familiarity with the requirements for operational readiness, this patient was able to resume his aviation career, albeit in a different capacity compatible with his condition.Chung CY, Lytle ME, Clemente Fuentes RW. A case of posterior nutcracker syndrome revealed in the aerospace environment. Aerosp Med Hum Perform. 2021; 92(1):5456.

Physical and Chemical Enhancement of and Adaptive Resistance to Irreversible Electroporation of Pancreatic Cancer.

Irreversible electroporation (IRE) can be used to treat cancer by electrical pulses, with advantages over traditional thermal approaches. Here we assess for the first time the IRE response of pancreatic cancer, one of the deadliest forms of cancer, both in vitro and in vivo. We demonstrate that both established and primary cancer cell lines can be destroyed by IRE, but with differential susceptibility and thresholds. We further demonstrate in vitro that viability for a given IRE dose can vary with the local chemistry as outcomes were shown to depend on suspending medium and reduction of glucose in the media significantly improved IRE destruction. Data here also demonstrate that repeated IRE treatments can lead to adaptive resistance in pancreatic carcinoma cells thereby reducing subsequent treatment efficacy. In addition, we demonstrate that physical enhancement of IRE, by re-arranging the pulse sequences without increasing the electrical energy delivered, achieve reduced viability in vitro and decreased tumor growth in an in vivo xenograft model. Together, these results show that IRE can destroy pancreatic cancer in vitro and in vivo, that there are both chemical and physical enhancements that can improve tumor destruction, and that one should guard against adaptive resistance when performing repeated treatments.

Exercise-induced hypoalgesia in women with varying levels of menstrual pain.

BACKGROUND AND AIMS: Exercise-induced hypoalgesia (EIH) is a well-established phenomenon in pain-free individuals that describes a decrease in pain sensitivity after an acute bout of exercise. The EIH response has been demonstrated to be sub-optimal in the presence of persisting pain. Menstrual pain is a common recurrent painful problem with many women experiencing high levels of pain each cycle. However, the EIH response has not been examined in a cohort of women with high levels of menstrual pain. This research aimed to examine whether EIH manifests differently in women with varying levels of menstrual pain. The primary hypothesis was that women with high levels of menstrual pain would demonstrate compromised EIH. Secondary aims were to explore relationships between EIH and emotional state, sleep quality, body mass index (BMI) or physical activity levels. METHODS: Pressure pain thresholds (PPT) were measured in 64 participants using a digital handheld algometer before and after a submaximal isometric-handgrip exercise. EIH index was compared between low (VAS 0-3), moderate (VAS 4-7) and high (VAS 8-10) pain groups, using a linear mixed model analysis with participant as a random effect, and site, menstrual pain category and the interaction between the two, as fixed effects. RESULTS: EIH was consistently induced in all groups. However, there was no statistically significant difference between the pain groups for EIH index (p=0.835) or for any co-variates (p>0.05). CONCLUSIONS: EIH was not found to differ between women who report regular low, moderate or high levels of menstrual pain, when measured at a point in their menstrual cycle when they are pain free. IMPLICATIONS: This study provides insight that EIH does not vary in women with differing levels of menstrual pain when they are not currently experiencing pain. The current findings indicate that, although menstrual pain can involve regular episodes of high pain levels, it may not be associated with the same central nervous system dysfunctions as seen in sustained chronic pain conditions.

α-Catenin homodimers are recruited to phosphoinositide-activated membranes to promote adhesion.

A unique feature of α-catenin localized outside the cadherin-catenin complex is its capacity to form homodimers, but the subcellular localization and functions of this form of α-catenin remain incompletely understood. We identified a cadherin-free form of α-catenin that is recruited to the leading edge of migrating cells in a phosphatidylinositol 3-kinase-dependent manner. Surface plasmon resonance analysis shows that α-catenin homodimers, but not monomers, selectively bind phosphatidylinositol-3,4,5-trisphosphate-containing lipid vesicles with high affinity, where three basic residues, K488, K493, and R496, contribute to binding. Chemical-induced dimerization of α-catenin containing a synthetic dimerization domain promotes its accumulation within lamellipodia and elaboration of protrusions with extended filopodia, which are attenuated in the α-cateninKKR<3A mutant. Cells restored with a full-length, natively homodimerizing form of α-cateninKKR<3A display reduced membrane recruitment, altered epithelial sheet migrations, and weaker cell-cell adhesion compared with WT α-catenin. These findings show that α-catenin homodimers are recruited to phosphoinositide-activated membranes to promote adhesion and migration, suggesting that phosphoinositide binding may be a defining feature of α-catenin function outside the cadherin-catenin complex.

Overview of hepatitis C and skin.

Hepatitis C (HCV) is the most common cause of chronic liver disease and hepatocellular carcinoma, as well as the leading indication for liver transplantation in the Western world. For many patients, cutaneous manifestations may be the only, the earliest, or the most apparent sign of the underlying infection. The dermatologic manifestations of HCV infection are reviewed.

Neuroprotection Mediated through GluN2C-Containing N-methyl-D-aspartate (NMDA) Receptors Following Ischemia.

Post-ischemic activation of NMDA receptors (NMDARs) has been linked to NMDAR subunit-specific signaling that mediates pro-survival or pro-death activity. Although extensive studies have been performed to characterize the role of GluN2A and GluN2B following ischemia, there is less understanding regarding the regulation of GluN2C. Here, we show that GluN2C expression is increased in acute hippocampal slices in response to ischemia. Strikingly, GluN2C knockout mice, following global cerebral ischemia, exhibit greater neuronal death in the CA1 area of the hippocampus and reduced spatial working memory compared to wild-type mice. Moreover, we find that GluN2C-expressing hippocampal neurons show marked resistance to NMDA-induced toxicity and reduced calcium influx. Using both in vivo and in vitro experimental models of ischemia, we demonstrate a neuroprotective role of GluN2C, suggesting a mechanism by which GluN2C is upregulated to promote neuronal survival following ischemia. These results may provide insights into development of NMDAR subunit-specific therapeutic strategies to protect neurons from excitotoxicity.

Genome Sequences of Gordonia Bacteriophages Obliviate, UmaThurman, and Guacamole.

We describe three newly isolated phages-Obliviate, UmaThurman, and Guacamole-that infect Gordonia terrae 3612. The three genomes are related to one another but are not closely related to other previously sequenced phages or prophages. The three phages are predicted to use integration-dependent immunity systems as described in several mycobacteriophages.

Early detection of thick melanomas in the United States: beware of the nodular subtype.

BACKGROUND: Incidence and mortality of melanoma in the United States have risen steeply. Part of this mortality increase may be related to late detection of biologically aggressive nodular melanomas. We determined trends in distribution of thin and thick melanoma, with emphasis on the histopathologic subtype nodular melanoma. METHODS: Surveillance, Epidemiology, and End Results melanoma incidence data for whites were obtained for 1988 through 1999 and stratified according to histologic subtype: lentigo maligna melanoma, superficial spreading melanoma, nodular melanoma, other, and not otherwise specified (NOS); thickness: 0-0.99 mm, 1.00 mm-1.99 mm, and > or =2.0 mm; patient age (0-49, 50+); gender; and year (1988-1991, 1992-1995, 1996-1999). Comparison of tumor thickness between strata was defined by year of diagnosis, sex, age, and histologic subtype. RESULTS: The number of new melanoma cases in a 3-year period increased 60% from 1988-1991 (n = 9132) to 1996-1999 (n = 14 575). The proportion of thick melanomas (> or =2 mm) remained relatively stable during the 12 study years. Nodular melanoma comprised 9% of all recorded cases but 34% of melanomas 2 mm or larger, including melanoma not otherwise specified (NOS), and nearly 50% of all melanomas 2 mm or larger when NOS cases were excluded. In contrast, superficial spreading melanoma was almost uniformly diagnosed as an early tumor, mostly (77%) presenting as thin melanoma (<1 mm) and with only 7% presenting as thick melanoma (> or =2 mm). CONCLUSIONS: A substantial number of thick melanomas in the United States are of the nodular subtype, and median thickness of nodular melanoma has not changed during the 12 years of study. New strategies are needed to decrease the incidence of thick melanoma in the United States.

Comparative analysis of the neurovascular injury and functional outcomes in experimental stroke models in diabetic Goto-Kakizaki rats.

Diabetes worsens functional outcome and is associated with greater hemorrhagic transformation (HT) after ischemic stroke. We have shown that diabetic Goto-Kakizaki (GK) rats develop greater HT and neurological deficit despite smaller infarcts after transient middle cerebral artery occlusion (MCAO) with the suture model. However, the impact of (1) the duration of ischemia/reperfusion (I/R); (2) the method of ischemia; and (3) acute glycemic control on neurovascular injury and functional outcome in diabetic stroke remained unanswered. Wistar and GK rats were subjected to variable MCAO by suture or embolus occlusion. A group of GK rats were treated with insulin or metformin before stroke with suture occlusion. In all groups, infarct size, edema, HT occurrence and severity, and functional outcome were measured. Infarct size at 24h was smaller in GK rats with both suture and embolic MCAO, but expanded with longer reperfusion period. Edema and HT were increased in GK rats after 90min and 3h occlusion with the suture model, but not in the embolic MCAO. Neurological deficit was greater in diabetic rats. These findings suggest that diabetes accelerates the development of HT and amplifies vascular damage in the suture model where blood flow is rapidly reestablished. Acute metformin treatment worsened the infarct size, HT, and behavior outcome, whereas insulin treatment showed a protective effect. These results suggest that the impact of ischemia/reperfusion on neurovascular injury and functional outcome especially in disease models needs to be fully characterized using different models of stroke to model the human condition.