Evaluation of Intraoperative Anastomotic Patency with Angiography in Microsurgical Breast Reconstruction.

Background: Microsurgical breast reconstruction is one of the most challenging, yet rewarding procedures performed by plastic surgeons. Several measures are taken to ensure safe elevation of the flap, preparation of recipient vessels, microvascular anastomosis, and flap inset. Reestablishing proper blood flow to the flap tissue after microvascular anastomosis is one of many critical steps for surgical success. Several measures to assess blood flow to the flap have been used; however, the use of indocyanine green angiography (ICGA) of the anastomosis in breast reconstruction has not been well documented. We present a series using ICGA for the evaluation of microvascular anastomosis success in breast reconstruction. Methods: Cases from patients who underwent microsurgical breast reconstruction between March 2022 and January 2023 and who had intraoperative ICGA were retrospectively reviewed. We compared the intraoperative findings on ICGA to flap success. Results: Sixteen patients underwent bilateral deep inferior epigastric perforator flap reconstruction with intraoperative ICGA of the microvascular anastomosis, constituting 32 deep inferior epigastric perforator flaps. The ICGA demonstrated return of blood flow in all the flaps after microvascular anastomosis and no flap loss in our sample population. Nine flaps required additional drainage using the superficial inferior epigastric vein, and the superficial circumflex vein was used for additional drainage in one of the flaps. Conclusions: The use of intraoperative ICGA provides reliable visual feedback regarding the patency and direction of the blood flow through the microvascular anastomosed vessels. ICGA can be used as an additional tool in the plastic surgeon's armamentarium for successful breast reconstruction.

Cervical Necrotizing Fasciitis, Diagnosis and Treatment of a Rare Life-Threatening Infection.

IMPORTANCE: Necrotizing fasciitis is a relatively uncommon and potentially life-threatening soft tissue infection, with morbidity and mortality approaching 25% to 35%, even with optimal treatment. The challenge of diagnosis for necrotizing soft tissue infections (NSTIs) is their rarity, with the incidence of approximately 1000 cases annually in the United States. Given the rapid progression of disease and its similar presentation to more benign processes, early and definitive diagnosis is imperative. FINDINGS: Signs and symptoms of NSTIs in the early stages are virtually indistinguishable from those seen with abscesses and cellulitis, making definitive diagnosis difficult. The clinical presentation will depend on the pathogen and its virulence factors which ultimately determine the area and depth of invasion into tissue. There are multiple laboratory value scoring systems that have been developed to support the diagnosis of an NSTI. The scoring system with the highest positive (92%) and negative (96%) predictive value is the laboratory risk indicator for necrotizing fasciitis (LRINEC). The score is determined by 6 serologic markers: C-reactive protein (CRP), total white blood cell (WBC) count, hemoglobin, sodium, creatinine, and glucose. A score ≥ 6 is a relatively specific indicator of necrotizing fasciitis (specificity 83.8%), but a score <6 is not sensitive (59.2%) enough to rule out necrotizing fasciitis. In terms of imaging, computed tomography (CT) imaging, while more sensitive (80%) than plain radiography in detecting abnormalities, is just as nonspecific. Computed tomography imaging of NSTIs demonstrates fascial thickening (with potential fat stranding), edema, subcutaneous gas, and abscess formation. Magnetic resonance imaging (MRI) has demonstrated sensitivity of 100% and specificity of 86%, though MRI may not show early cases of fascial involvement of necrotizing fasciitis. CONCLUSIONS AND RELEVANCE: Necrotizing soft tissue infections are rapidly progressive and potentially fatal infections that require a high index of clinical suspicion to promptly diagnose and aggressive surgical debridement of affected tissue in order to ensure optimal outcomes.Prompt surgical and infectious disease consultation is necessary for the treatment and management of these patients. While imaging is useful for further characterization, it should not delay surgical consultation. Necrotizing soft tissue infection remains a clinical diagnosis, although plain radiography, CT imaging, and ultrasound can provide useful clues. In general, the management of these patients should include rapid diagnosis, using a combination of clinical suspicion, laboratory data (LRINEC score), and imaging (MRI being the recommended imaging modality), prompt infectious disease and surgical consultation, surgical debridement, and delayed reconstruction. Laboratory findings that can more strongly suggest a diagnosis of NSTI include elevated CRP, elevated WBC, low hemoglobin, decreased sodium, and increased creatinine. Imaging findings include fascial thickening (with potential fat stranding), edema, subcutaneous gas, and abscess formation. Broad-spectrum antibiotics should be started in all cases of suspected NSTI. Surgical debridement, however, remains the lynchpin for treatment of cervical necrotizing fasciitis.

Unilateral Corporal Cavernosum Partial Thrombosis: A Challenging Presentation and Management.

A 29-year-old African American male presented to our emergency department with a 5-day history of perineal pain and tender swelling of the left perineal body with no evidence of trauma. Physical examination revealed a firm left proximal corpus cavernosum body. Laboratory tests were within normal limits. Pelvic MRI confirmed the presence of a large left-sided unilateral corporal cavernosum partial thrombosis filling the proximal third of the left corpus cavernosum. The patient was managed conservatively with anticoagulation, pain control, and pelvic rest. At 3-month follow-up, perineal imaging showed reduction of thrombus size and resolution of pain and swelling. At 6-month follow-up, a penile ultrasound demonstrated almost complete resolution of the thrombus.

Imaging findings of a twin male neonate with megacystis microcolon intestinal hypoperistalsis syndrome.

Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS) is a rare congenital defect of the bowel and bladder that is especially rare in males. We present a case of MMIHS in a male nonidentical twin neonate who presented with abdominal distention, urinary retention, and hypoplastic bowel. The voiding cystourethrogram included in this report displays rare image of MMIHS in a male urogenital system. The constellation of clinical and imaging findings presented in this case are characteristic of MMIHS and may aid the early diagnosis of male neonates affected by this disease.

Virtual Surgical Planning in Oral and Maxillofacial Surgery.

Virtual surgical planning provides the ability to visualize the patient anatomy and pathologic condition, establish pertinent reference points, and simulate osteotomies and reconstruction design in advance of the surgery. Virtual surgical planning is also an ideal environment for improved communication between the patient and the surgical team.

Complete robotic repair of a renal artery aneurysm.

Although the majority of renal artery aneurysms require only observation, those that require treatment have been addressed primarily surgically or endovascularly. We report a case of surgical resection of a large, symptomatic renal artery aneurysm from an entirely robotic approach.

Circadian Dysfunction Induces Leptin Resistance in Mice.

Circadian disruption is associated with obesity, implicating the central clock in body weight control. Our comprehensive screen of wild-type and three circadian mutant mouse models, with or without chronic jet lag, shows that distinct genetic and physiologic interventions differentially disrupt overall energy homeostasis and Leptin signaling. We found that BMAL1/CLOCK generates circadian rhythm of C/EBPα-mediated leptin transcription in adipose. Per and Cry mutant mice show similar disruption of peripheral clock and deregulation of leptin in fat, but opposite body weight and composition phenotypes that correlate with their distinct patterns of POMC neuron deregulation in the arcuate nucleus. Chronic jet lag is sufficient to disrupt the endogenous adipose clock and also induce central Leptin resistance in wild-type mice. Thus, coupling of the central and peripheral clocks controls Leptin endocrine feedback homeostasis. We propose that Leptin resistance, a hallmark of obesity in humans, plays a key role in circadian dysfunction-induced obesity and metabolic syndromes.

Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis.

Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics. Specifically, ADI with either 12 kDa or 20 kDa PEG attachments on 33% of the primary amines retained about 60% or 48% of enzyme activity, respectively; the Km and pH profiles were nearly unchanged; IC50 values were diminished by less than 30%; while stability studies demonstrated full retention of activity at 4 degrees C for 5 months. A comparison of the enzymatic properties of a second ADI from Pseudomonas putida illustrated the superior characteristics of the M. arthritidis ADI enzyme.

Structure-function engineering of interferon-beta-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation.

Recombinant interferon-beta-1b (IFN-beta-1b) is used clinically in the treatment of multiple sclerosis. In common with many biological ligands, IFN-beta-1b exhibits a relatively short serum half-life, and bioavailability may be further diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increase the blood residency time of protein therapeutics, there is a further requisite for molecular engineering approaches to also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins. We investigated these five parameters of recombinant human IFN-beta-1b in over 20 site-selective mono-PEGylated or multi-PEGylated IFN-beta-1b bioconjugates. Primary amines were modified by single or multiple attachments of poly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternate approaches, site-directed mutagenesis was independently employed in the construction of designed IFN-beta-1b variants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugate preparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and a comparison of the structural and functional properties of the IFN-beta-1b proteins and their PEGylated counterparts was conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sites of the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferation bioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusion HPLC, and immunoassays demonstrated that the functional activities of the designed IFN-beta-1b conjugates were maintained, while the formation of soluble or insoluble aggregates of IFN-beta-1b was ameliorated. Immunogenicity and pharmacokinetic studies of selected PEGylated IFN-beta-1b compounds in mice and rats demonstrated both diminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. The results demonstrate the capacity of this macromolecular engineering strategy to address both pharmacological and formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN-beta-1b, were further investigated in formulation optimization and biological studies.

Tailoring structure-function and pharmacokinetic properties of single-chain Fv proteins by site-specific PEGylation.

The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V(L)-linker-V(H) and V(H)-linker-V(L). High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Independent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.

Methanocalculus taiwanensis sp. nov., isolated from an estuarine environment.

Two novel hydrogenotrophic methanogens, designated strains P2F9704aT and P2F9705, were isolated from an estuary in Eriln Shi, Taiwan. The cells of strain P2F9704aT were non-motile, irregular cocci 0.9-1.4 microm in diameter. They stained gram-negative. The cells catabolized formate and H2+CO2 to produce methane, but did not utilize acetate, methanol, trimethylamine, ethanol or secondary alcohols as methanogenic substrates. The optimal growth parameters for strain P2F9704aT were pH 6.7, 37 degrees C and 0.5% NaCl. Acetate was required for cell growth even though it was not a substrate for methanogenesis. The trace element tungsten was not required but slightly stimulated the growth of strain P2F9704aT. However, tungsten extended the growth ranges relating to temperature, pH and salt. The sequences of the 16S rRNA genes of strains P2F9704aT and P2F9705 were nearly identical and possessed 99.1 and 98.5% similarity to the genes of Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, strain P2F9704aT possessed 14 and 12% DNA relatedness with respect to Methanocalculus pumilus and Methanocalculus halotolerans, respectively. In addition, the optimal salt concentrations, the cellular protein profiles and the molecular masses of surface-layer protein subunits of strain P2F9704aT were different from those of the other two known Methanocalculus species. On the basis of these observations, it is proposed that these two organisms should be placed in a new species, namely Methanocalculus taiwanensis. The type strain is P2F9704aT (= OCM 671T = CCRC 16182T = DSM 14663T).