Vanucizumab mode of action: Serial biomarkers in plasma,tumor, and skin-wound-healing biopsies

Vanucizumab is a novel bispecific antibody inhibiting vascular endothelial growth factor (VEGF-A) and angiopoietin-2 (Ang-2) that demonstrated safety and anti-tumor activity in part I of a phase I study of 42 patients with advanced solid tumors. Part II evaluated the pharmacodynamic effects of vanucizumab 30 or 15 mg/kg every 2 weeks in 32 patients. Serial plasma samples, paired tumor, and skin-wound-healing biopsies were taken over 29 days to evaluate angiogenic markers. Vanucizumab was associated with marked post-infusion reductions in circulating unbound VEGF-A and Ang-2. By day 29, tumor samples revealed mean reductions in density of microvessels (−32.2%), proliferating vessels (−47.9%) and Ang-2 positive vessels (−62.5%). Skin biopsies showed a mean reduction in density of microvessels (−49.0%) and proliferating vessels (−25.7%). Gene expression profiling of tumor samples implied recruitment and potential activation of lymphocytes. Biopsies were safely conducted. Vanucizumab demonstrated a consistent biological effect on vascular-related biomarkers, confirming proof of concept. Skin-wound-healing biopsies were a valuable surrogate for studying angiogenesis-related mechanisms.     

Serial cross-sectional estimation of vaccine-and infection-induced SARS-CoV-2 seroprevalence in British Columbia,Canada

Background:The evolving proportion of the population considered immunologically naive versus primed for more efficient immune memory response to SARS-CoV-2 has implications for risk assessment. We sought to chronicle vaccine- and infection-induced seroprevalence across the first 7 waves of the COVID-19 pandemic in British Columbia, Canada.Methods:During 8 cross-sectional serosurveys conducted between March 2020 and August 2022, we obtained anonymized residual sera from children and adults who attended an outpatient laboratory network in the Lower Mainland (Greater Vancouver and Fraser Valley). We used at least 3 immunoassays per serosurvey to detect SARS-CoV-2 spike and nucleocapsid antibodies. We assessed any seroprevalence (vaccineor infection-induced, or both), defined by positivity on any 2 assays, and infection-induced seroprevalence, also defined by dual-assay positivity but requiring both antinucleocapsid and antispike detection. We used estimates of infection-induced seroprevalence to explore underascertainment of infections by surveillance case reports.Results:By January 2021, we estimated that any seroprevalence remained less than 5%, increasing with vaccine rollout to 56% by May–June 2021, 83% by September–October 2021 and 95% by March 2022. Infection-induced seroprevalence remained less than 15% through September–October 2021, increasing across Omicron waves to 42% by March 2022 and 61% by July–August 2022. By August 2022, 70%–80% of children younger than 20 years and 60%–70% of adults aged 20–59 years had been infected, but fewer than half of adults aged 60 years and older had been infected. Compared with estimates of infection-induced seroprevalence, surveillance case reports underestimated infections 12-fold between September 2021 and March 2022 and 92-fold between March 2022 and August 2022.Interpretation:By August 2022, most children and adults younger than 60 years had evidence of both SARS-CoV-2 vaccination and infection. As previous evidence suggests that a history of both exposures may induce stronger, more durable hybrid immunity than either exposure alone, older adults — who have the lowest infection rates but highest risk of severe outcomes — continue to warrant prioritized vaccination.

The British Columbia Centre for Disease Control (BCCDC) has a long-established serosurvey protocol to monitor population susceptibility to emerging or re-emerging respiratory viruses. The approach was first deployed during the influenza A (H1N1) pandemic in 2009 to monitor changes in seroprevalence across successive pandemic waves and the mass vaccination campaign.^1–7 The methodology is predicated upon serial cross-sectional convenience sampling of anonymized residual sera from children and adults of all ages in the most populated Lower Mainland region of BC.^8,9Adapting this protocol, the BCCDC launched its first SARS-CoV-2 serosurvey in March 2020, just before the World Health Organization’s declaration of the COVID-19 pandemic. ¹⁰ Baseline assessment was followed by additional serosurveys that spanned the time from mRNA vaccine availability in mid-December 2020, through 7 pandemic waves associated with multiple variants of concern to August 2022 (Figure 1).^11–13 Using these serosurveys, we sought to track the evolving proportion of the population that remained immunologically naive and, thus, fully susceptible to COVID-19, versus the evolving proportion that was immunologically primed (through vaccination or infection) and, thus, poised for more efficient memory response in mitigating the risk of SARS-CoV-2. Recognizing the spectrum of illness, including asymptomatic or mild infections, and variable diagnostic access, case identification and reporting, we also used estimates of infection-induced seroprevalence to explore the potential underascertainment of infections by surveillance case reports.Open in a separate windowFigure 1:Provincial surveillance case reports to the British Columbia Centre for Disease Control (BCCDC) by epidemiological week from January 2020 to September 2022, with timing of serosurveys and select public health measures, in BC, Canada. We group case tallies by epidemiological week (7-d period) as per standard surveillance methods for comparing data by period from year to year. Epidemic waves are enumerated sequentially and are displayed with the predominant variant of concern (VOC). Publicly funded access to nucleic acid amplification tests (NAATs) or rapid antigen tests (RATs) is displayed below the X-axis. For details on public health measures, vaccines, schedules and coverage estimates, see Appendix 1, Supplementary Material 1, available at www.cmaj.ca/lookup/doi/10.1503/cmaj.221335/tab-related-content. *Nonessential travel discouraged, health care service delivery adjusted, public gatherings > 50 people prohibited. Provincial state of emergency declared. †Interactions limited to households or “core bubble” (immediate family or those in same dwelling) or to a maximum of 2 other people if living alone. ‡Dine-in food services and indoor fitness activities banned, only essential travel permitted. §Gradual return to gatherings, recreational travel, in-person work, which was interrupted by the fourth wave. ¶Indoor and personal gatherings limited, 50% capacity limit at venues of > 1000 people, sports tournaments paused. Social restrictions lifted during epidemiological week 7, 2022. **Mask mandates lifted during epidemiological week 10, 2022. ††The first 2 spike-based mRNA vaccine formulations were authorized during epidemiological weeks 50 and 52, 2020, respectively, with mRNA vaccines comprising most doses (> 90%) administered in BC and Canada across the pandemic. In epidemiological week 8, 2021, a chimpanzee adenoviral-vectored (ChAdOx1) vaccine was also authorized. ‡‡Vaccines (mRNA) initially deployed to high-risk individuals, including residents and staff of long-term care and assisted-living facilities, essential visitors within those settings and health care workers. §§Community-based vaccine roll-out, prioritized by age, beginning with the oldest adults in mid-March 2021. Access to booster doses followed similar prioritization sequence, inclusive of clinically extremely vulnerable individuals of any age. ¶¶Single-dose vaccine card required for entry into social and recreational settings starting in epidemiological week 37, 2021; 2-dose cards were required beginning in epidemiological week 43, 2021. Vaccine cards were ultimately repealed in epidemiological week 14, 2022.     

Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation

Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass‐to‐ethanol pipeline. Here, the feasibility of scaling‐up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H₂O₂/g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose‐ and xylose‐utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. 2012; 109:922–931. © 2011 Wiley Periodicals, Inc.     

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.     

<Emphasis Type="Italic">Rubus rosaefolius</Emphasis> Extract as a Natural Preservative Candidate in Topical Formulations

Even though the synthetic preservatives may offer a high antimicrobial efficacy, they are commonly related to adverse reactions and regarded as having potentially harmful effects caused by chronic consumption. The development of natural preservatives provides a way of reducing the amount of synthetic preservatives normally used in pharmaceutical and cosmetic preparations. In addition, these agents have less toxic effects and represent a possible natural and safer alternative of the preservatives. The purpose of this research was to evaluate the Rubus rosaefolius Smith extract efficiency as a natural preservative in base formulations. Of the extract, 0.2% (w/w) was assayed for its effectiveness of antimicrobial protection in two different base formulations (emulsion and gel). The microbial challenge test was performed following the standard procedures proposed by The United States Pharmacopoeia 33nd, European Pharmacopoeia 6th, Japanese Pharmacopoeia 15th, and the Cosmetics, Toiletries, and Fragrance Association using standardized microorganisms. The results demonstrated that R. rosaefolius extract at the studied concentration reduced the bacterial inocula, satisfying the criterion in all formulations, even though it was not able to present an effective preservative behavior against fungi. Thus, the investigation of new natural substances with preservative properties that could be applied in pharmaceutical and cosmetic products is relevant due to the possibility of substituting or decreasing the concentration of synthetic preservatives, providing a way for the development of safer formulas for the use of consumers.     

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).     

Has Ombrëdanne's method of hypospadic urethra reconstruction been ignored with reason?

From January 1970 to December 1979 inclusive, 193 boys (aged 2 to 16) underwent surgery for distal hypospadia using Ombredanne's method at the Department of Pediatric Surgery University Hospital Center Rijeka and at the Department of Pediatric Surgery Zagreb. Follow-up period was 7 to 20 years (mean 13.4). 20 (10.36%) subjects had post-operative organic complications and 15 (7.77%) of them required surgical correction. According to these findings, the success rate using Ombredanne's method of reconstruction of the hypospadic urethra in no way lags behind the success rate using MAGPI and Mathieu's methods as well as Preputial island flap urethroplasty for analogous cases. Out of 193 subjects who underwent surgery, 80 (41.45%) of those who were sexually mature and had normal psychosexual development were questioned. In this sample, 75 (93.75%) were satisfied with the post-operative appearance of the penis while only 5 (6.25%) were dissatisfied, 3 of which had hypoplastic penis. In 78 (97.50%) subjects questioned, the post-operative urinary squirt was normal and two of them had weak urinary squirt (2.50%), due to meatal stenosis. In conclusion, Ombredanne's method of reconstruction of the urethra in boys with distal hypospadia is equally successful as other methods used for this purpose.     

Synthesis and anti-aromatase activity of some new steroidal D-lactones

Starting from D-seco derivatives of 5-androstene 1-3, the D-homo lactones, 4 and 5, were synthesized. By the Oppenauer oxidation and/or by dehydration of 4 and 5 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), the corresponding D-lactones 6-12 were obtained. The structures of 6 and 10 were unambiguously proved by the appropriate X-ray structural analysis. Anti-aromatase assay showed that tested compounds possess inhibition potency, however, two to four times smaller (IC50 from 0.2 to 0.7 microM, respectively) in comparison to aminoglutethimide (AG).