Fertility and Pregnancy-Related Issues in Young BRCA Carriers With Breast Cancer.

Approximately 10% to 15% of breast cancer cases in young women are diagnosed in patients harbouring germline (g) pathogenic or likely pathogenic variants (PVs) in the BReast CAncer 1 (BRCA1) or BReast CAncer 2 (BRCA2) genes. Preclinical and clinical studies showed a potential negative effect of germline BRCA1/2 (gBRCA1/2) PVs on ovarian reserve and reproductive potential, even before starting anticancer therapies. The aim of this article is to summarize the current literature on the fertility potential of young gBRCA1/2 PVs carriers with breast cancer and the risk of gonadotoxicity associated with anticancer treatments. Moreover, we describe the available evidence on the efficacy of fertility preservation techniques in young gBRCA1/2 PVs carriers and the safety data on having a pregnancy after breast cancer treatment.

A Systematic Review of the Gonadotoxicity of Osteosarcoma and Ewing's Sarcoma Chemotherapies in Postpubertal Females and Males.

Data on gonadotoxicity of chemotherapies are essential to better counsel young females and males about the risk of infertility and to better indicate fertility preservation measures before cancer therapies. However, such data have not recently been reviewed for bone cancer. Therefore, a systematic literature search was conducted considering papers published since 2000. This study is part of the FertiTOX® project, which aims to improve the lack of data regarding gonadotoxicity of cancer therapies to enable more accurate counseling regarding fertility preservation. Only relapse-free women and men were included. Gonadotoxic therapy-induced suspected infertility was defined as very low anti-mullerian hormone, high gonadotropin concentration, amenorrhea, oligomenorrhea, azoospermia, or oligozoospermia. The quality of the individual studies was assessed using the Newcastle-Ottawa Scale (NOS). In total, 11 out of 831 studies were included in the review. Suspected infertility was found in 10/190 (5.1%, range 0%-66%) of female patients with osteosarcoma (six studies), in 24/46 (52.2%, range 46%-100%) of male patients with osteosarcoma (three studies), in 18/138 (13.0%, range 3%-18%) of female patients with Ewing's sarcoma (three studies), and in 34/38 (89.5%) of male patients with Ewing's sarcoma (one study). A risk calculation in relation to specific chemotherapies was not possible. Risk of suspected infertility tends to be higher in Ewing's sarcoma in which all patients received chemotherapies with alkylating agents. Two of the 11 included studies received a high NOS quality score, whereas the remaining nine studies received a low quality score, mainly because of the lack of a comparator group. Published data are too limited for precise estimation of the gonadotoxicity. However, data indicate clinically relevant risk for infertility, supporting counseling patients before chemotherapy about fertility preservation measures.

Evaluation of the Gonadotoxicity of Cancer Therapies to Improve Counseling of Patients About Fertility and Fertility Preservation Measures: Protocol for a Retrospective Systematic Data Analysis and a Prospective Cohort Study.

BACKGROUND: Cytotoxic treatments such as chemo- and radiotherapy and immune therapies are required in cancer diseases. These therapies have the potential to cure patients but may also have an impact on gonadal function and, therefore, on fertility. Consequently, fertility preservation treatments such as freezing of gametes and gonadal tissue might be required. However, as detailed data about the necessity to perform fertility preservation treatment are very limited, this study was designed to fill this data gap. OBJECTIVE: Primary objective of this study is to analyze the impact of cancer therapies and chemotherapies on the ovarian reserve and sperm quality. Secondary objectives are to analyze the (1) impact of cancer therapies and chemotherapies on other fertility parameters and (2) probability of undergoing fertility preservation treatments in relation to specific cancer diseases and treatment protocols and the probability to use the frozen gametes and gonadal tissue to achieve pregnancies. METHODS: First, previously published studies on the gonadotoxicity of chemo- and radiotherapies among patients with cancer will be systematically analyzed. Second, a prospective cohort study set up by approximately 70 centers in Germany, Switzerland, and Austria will collect the following data: ovarian function by analyzing anti-Müllerian hormone (AMH) concentrations and testicular function by analyzing sperm parameters and total testosterone immediately before and around 1 year after gonadotoxic therapies (short-term fertility). A follow-up of these fertility parameters, including history of conceptions, will be performed 5 and 10 years after gonadotoxic therapies (long-term fertility). Additionally, the proportion of patients undergoing fertility-preserving procedures, their satisfaction with these procedures, and the amount of gametes and gonadal tissue and the children achieved by using the frozen material will be analyzed. Third, the data will be merged to create the internet-based data platform FertiTOX. The platform will be structured in accordance with the ICD (International Classification of Diseases) classification of cancer diseases and will be easily be accessible using a specific App. RESULTS: Several funding bodies have funded this study. Ten systematic reviews are in progress and the first one has been accepted for publication. All Swiss and many German and Austrian ethics committees have provided their approval for the prospective cohort study. The study registry has been set up, and a study website has been created. In total, 50 infertility centers have already been prepared for data collection, which started on December 1, 2023. CONCLUSIONS: The study can be expected to bridge the data gap regarding the gonadotoxicity of cancer therapies to better counsel patients about their infertility risk and their need to undergo fertility preservation procedures. Initial data are expected to be uploaded on the FertiTOX platform in 2026. TRIAL REGISTRATION: ClinicalTrials.gov NCT05885048; https://clinicaltrials.gov/study/NCT05885048. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/51145.

Measuring Quality of Life: Incorporating Objectively Measurable Parameters within the Cross-Sectional Bern Cohort Study 2014 (BeCS-14).

Up until now, the measurement of Quality of Life (QoL) was based on validated subjective rating tools rather than objective measurement. To become more independent of the self-assessment of probands, a way to objectively measure QoL should be found. A monocenter, cross-sectional, observational, non-interventional trial was performed from 2012 to 2014 at Inselspital Bern to evaluate the bio-functional status (BFS), a complex, generic, non-invasive, sex- and age-validated assessment tool, in a wide range of areas. A standardized battery of assessments was performed on 464 females and 166 males, ages 18 to 65 (n = 630). In addition to the survey of the BFS, participants replied-among others-to the validated questionnaire SF-36 for health-related QoL (n = 447, subgroup 1). Since the accepted cut-off value for BFA calculation is age ≥ 35 years, subgroup 2 included 227 subjects (all participants aged ≥ 35 years out of subgroup 1). In order to be able to compare the eight SF-36 subscales to BFS parameters, a comparable score set of single BFS items had to be constructed. Subsequently, we aimed to statistically identify BFS item combinations that best represented each SF-36 subscale. All eight SF-36 subscales were significantly represented by various different combinations of BFS items. A total of 24 single BFS items significantly correlated with SF-36 subscales, of which 15 were objective and nine were subjective. All eight SF-36 subscales were significantly represented by various different combinations of BFS items leading to stronger correlations (range five to nine BFS items), and overall, sex and age did not affect these associations, but in the SF-36 subscales 'bodily pain' (sex) and 'role limitations due to physical health problems' (age in men). To our knowledge, we are the first to correlate a validated set of 34 objective and 9 subjective parameters with subjectively evaluated SF-36 subscales. This first study on the objectifiability of the SF-36 questionnaire demonstrated that questions on quality of life can be answered independently of a subjective assessment by subjects in future scientific studies.

Progestogens for endometrial protection in combined menopausal hormone therapy: A systematic review.

Menopausal women with an intact uterus choosing estrogens for menopausal symptom relief require a progestogen for endometrial protection. The aim of this systematic review was to evaluate the risks of endometrial hyperplasia resp. malignancy with different progestogens used in combined MHT. Overall, 84 RCTs were included. We found that 1) most studies were done with NETA, followed by MPA, MP and DYD and LNG, 2) most progestogens were only available as oral formulations, 3) the most frequently studied progestogens (oral MP, DYD, MPA, oral and transdermal NETA, transdermal LNG) were assessed in continuously as well as in sequentially combined MHT regimens, 4) FDA endometrial safety criteria were only fulfilled for some progestogen formulations, 5) most studies demonstrated endometrial protection for the progestogen dose and time period examined. However, 6) study quality varied which should be taken into account, when choosing a combined MHT, especially if off-label-use is chosen.

A single fiber view of the nucleosome organization in eukaryotic chromatin.

Eukaryotic cells are thought to arrange nucleosomes into extended arrays with evenly spaced nucleosomes phased at genomic landmarks. Here we tested to what extent this stereotypic organization describes the nucleosome organization in Saccharomyces cerevisiae using Fiber-Seq, a long-read sequencing technique that maps entire nucleosome arrays on individual chromatin fibers in a high throughput manner. With each fiber coming from a different cell, Fiber-Seq uncovers cell-to-cell heterogeneity. The long reads reveal the nucleosome architecture even over repetitive DNA such as the ribosomal DNA repeats. The absolute nucleosome occupancy, a parameter that is difficult to obtain with conventional sequencing approaches, is a direct readout of Fiber-Seq. We document substantial deviations from the stereotypical nucleosome organization with unexpectedly long linker DNAs between nucleosomes, gene bodies missing entire nucleosomes, cell-to-cell heterogeneity in nucleosome occupancy, heterogeneous phasing of arrays and irregular nucleosome spacing. Nucleosome array structures are indistinguishable throughout the gene body and with respect to the direction of transcription arguing against transcription promoting array formation. Acute nucleosome depletion destroyed most of the array organization indicating that nucleosome remodelers cannot efficiently pack nucleosomes under those conditions. Given that nucleosomes are cis-regulatory elements, the cell-to-cell heterogeneity uncovered by Fiber-Seq provides much needed information to understand chromatin structure and function.

Optimization and validation of analytical affinity chromatography for the in-process monitoring and quantification of peptides containing a C-tag.

Antimicrobial peptides and proteins (AMPs) are promising alternatives to conventional antibiotics for the treatment of infections caused by multidrug-resistant bacteria. The production of recombinant AMPs is facilitated by platform technologies such as the C-tag, a sequence of four C-terminal amino acids that allows immunoaffinity capture and purification. However, the detection and quantification of such products throughout the manufacturing process is a significant challenge. We therefore used a design of experiments approach to optimize a novel high-throughput analytical immunoaffinity chromatography method for the accurate quantification of AMPs containing a C-tag, resulting in minimal analyte carryover (98.8 ± 0.1 % product elution). We then validated the method in accordance with International Conference on Harmonisation guideline Q2(R2). Validation confirmed that the method achieves high specificity, linearity, accuracy, and precision. We implemented in-process control and quantification throughout the manufacturing process, from cell lysis to the final purified product. We found that the lysate and acidic samples (pH < 2) can lead to deviations. However, following sample pretreatment, C-tag quantification reduced the error to ≤ 4 %, which is potentially superior to current non-specific quantification methods such as UV absorbance and colorimetry. Implementing this method for in-process control and quantification throughout the manufacturing process achieves the reliable assessment of product quantity and quality. This method also offers improvements over the product-specific enzyme-linked immunosorbent assay currently used for C-tagged products because it has a higher precision, accuracy and throughput, with a measurement time of 2.5 min per sample. Our analytical affinity chromatography method is therefore a valuable tool for the quantification of AMPs as part of a novel platform technology approach for C-tagged products.

Unveiling the secrets of adeno-associated virus: novel high-throughput approaches for the quantification of multiple serotypes.

Adeno-associated virus (AAV) vectors are among the most prominent viral vectors for in vivo gene therapy, and their investigation and development using high-throughput techniques have gained increasing interest. However, sample throughput remains a bottleneck in most analytical assays. In this study, we compared commonly used analytical methods for AAV genome titer, capsid titer, and transducing titer determination with advanced methods using AAV2, AAV5, and AAV8 as representative examples. For the determination of genomic titers, we evaluated the suitability of qPCR and four different digital PCR methods and assessed the respective advantages and limitations of each method. We found that both ELISA and bio-layer interferometry provide comparable capsid titers, with bio-layer interferometry reducing the workload and having a 2.8-fold higher linear measurement range. Determination of the transducing titer demonstrated that live-cell analysis required less manual effort compared with flow cytometry. Both techniques had a similar linear range of detection, and no statistically significant differences in transducing titers were observed. This study demonstrated that the use of advanced analytical methods provides faster and more robust results while simultaneously increasing sample throughput and reducing active bench work time.

An investigation of excipients for a stable Orf viral vector formulation.

The Orf virus (ORFV) is a promising candidate for vector vaccines as well as for immunomodulatory and oncolytic therapies. However, few publications are available on its infectivity degradation or on suitable additives for prolonging its viral stability. In this study, the non-supplemented ORFV itself showed a very high stability at storage temperatures up to 28 °C, with a linear titer loss of 0.10 log infectious particles per day at 4 °C over a period of five weeks. To prolong this inherent stability, thirty additives, i.e., detergents, sugars, proteins, salts, and buffers as well as amino acids, were tested for their time- and temperature-dependent influence on the ORFV infectivity. A stabilizing effect on the infectivity was identified for the addition of all tested proteins, i.e., gelatine, bovine serum albumin, and recombinant human serum albumin (rHSA), of several sugars, i.e., mannitol, galactose, sucrose, and trehalose, of amino acids, i.e., arginine and proline, of the detergent Pluronic F68, and of the salt Na2SO4. The infectivity preservation was especially pronounced for proteins in liquid and frozen formulations, sugars in frozen state, and arginine und Pluronic in liquid formulations at high storage temperatures (37 °C). The addition of 1% rHSA with and without 5% sucrose was evaluated as a very stable formulation with a high safety profile and economic validity at storage temperatures up to 28 °C. At increased temperatures, the supplementation with 200 mM arginine performed better than with rHSA. In summary, this comprehensive data provides different options for a stable ORFV formulation, considering temperature, storage time, economic aspects, and downstream processing integrity.

Evaluation of restricted access media for the purification of cell culture-derived Orf viruses.

Recently, multimodal chromatography using restricted access media (RAM) for the purification of nanoparticles, such as viruses has regained increasing attention. These chromatography resins combine size exclusion on the particle shell and adsorptive interaction within the core. Accordingly, smaller process-related impurities, for example, DNA and proteins, can be retained, while larger product viruses can pass unhindered. We evaluated a range of currently available RAM, differing in the shells' pore cut-off and the core chemistry, for the purification of a cell culture-derived clarified model virus, namely the Orf virus (ORFV). We examined impurity depletion and product recovery as relevant criteria for the evaluation of column performance, as well as scale-up robustness and regeneration potential for evaluating a multiple use application. The results indicate that some columns, for example, the Capto Core, enable both a high DNA and protein removal, while others, for example, the Monomix Core 60 (MC60), are more suitable for DNA depletion. Furthermore, column regeneration is facilitated by using columns with larger shell pores (5000 vs. 700 kDa) and weaker binding interactions (anion exchange vs. multimodal). According to these findings, the choice of RAM resins should be selected according to the respective feed sample composition and the planned number of application cycles.

Affinity and Pseudo-Affinity Membrane Chromatography for Viral Vector and Vaccine Purifications: A Review.

Several chromatographic approaches have been established over the last decades for the production of pharmaceutically relevant viruses. Due to the large size of these products compared to other biopharmaceuticals, e.g., proteins, convective flow media have proven to be superior to bead-based resins in terms of process productivity and column capacity. One representative of such convective flow materials is membranes, which can be modified to suit the particular operating principle and are also suitable for economical single-use applications. Among the different membrane variants, affinity surfaces allow for the most selective separation of the target molecule from other components in the feed solution, especially from host cell-derived DNA and proteins. A successful membrane affinity chromatography, however, requires the identification and implementation of ligands, which can be applied economically while at the same time being stable during the process and non-toxic in the case of any leaching. This review summarizes the current evaluation of membrane-based affinity purifications for viruses and virus-like particles, including traditional resin and monolith approaches and the advantages of membrane applications. An overview of potential affinity ligands is given, as well as considerations of suitable affinity platform technologies, e.g., for different virus serotypes, including a description of processes using pseudo-affinity matrices, such as sulfated cellulose membrane adsorbers.

Process intensification for the production of a C-tagged antimicrobial peptide in Escherichia coli - First steps toward a platform technology.

The production of antimicrobial peptides/proteins (AMPs) in sufficient quantities for clinical evaluation is challenging because complex peptides are unsuitable for chemical synthesis, natural sources have low yields, and heterologous systems often have low expression levels or require product-specific process adaptations. Here we describe the production of a complex AMP, the insect metalloproteinase inhibitor (IMPI), by adding a C-terminal C-tag to increase the yield compared to the unmodified peptide. We used a design of experiments approach for process intensification in Escherichia coli Rosetta-gami 2(DE3)pLysS cells and achieved a yield of 260 mg L-1, which is up to 30-fold higher than previously reported. The C-tag also enhanced product purity but had no effect on IMPI activity, making tag removal unnecessary and therefore simplifying process analytics and downstream processing. We have confirmed that the C-tag is compatible with the peptide and could form the basis of a platform technology for the expression, purification and detection of diverse AMPs produced in E. coli.

Impact of estrogens on resting energy expenditure: A systematic review.

The fear of weight gain is one of the main reasons for women not to initiate or to early discontinue hormonal contraception or menopausal hormone therapy. Resting energy expenditure is by far the largest component and the most important determinant of total energy expenditure. Given that low resting energy expenditure is a confirmed predictive factor for weight gain and consecutively for the development of obesity, research into the influence of sex steroids on resting energy expenditure is a particularly exciting area. The objective of this systematic review was to evaluate the effects of medication with natural and synthetic estrogens on resting energy expenditure in healthy normal weight and overweight women. Through complex systematic literature searches, a total of 10 studies were identified that investigated the effects of medication with estrogens on resting energy expenditure. Our results demonstrate that estrogen administration increases resting energy expenditure by up to +208 kcal per day in the context of contraception and by up to +222 kcal per day in the context of menopausal hormone therapy, suggesting a preventive effect of circulating estrogen levels and estrogen administration on weight gain and obesity development.

Recurrent Miscarriage: Diagnostic and Therapeutic Procedures. Guideline of the DGGG, OEGGG and SGGG (S2k-Level, AWMF Registry No. 015/050, May 2022).

Purpose The aim of this guideline is to standardize the diagnosis and therapy of recurrent miscarriage (RM) using evidence from the recent literature. This is done by using consistent definitions, objective evaluations and standardized treatment protocols. Methods When this guideline was compiled, special consideration was given to previous recommendations in prior versions of this guideline and the recommendations of the European Society of Human Reproduction and Embryology, the Royal College of Obstetricians and Gynecologists, the American College of Obstetricians and Gynecologists and the American Society for Reproductive Medicine, and a detailed individual search of the literature about the different topics was carried out. Recommendations Recommendations about the diagnostic and therapeutic procedures offered to couples with RM were developed based on the international literature. Special attention was paid to known risk factors such as chromosomal, anatomical, endocrinological, physiological coagulation, psychological, infectious and immune disorders. Recommendations were also developed for those cases where investigations are unable to find any abnormality (idiopathic RM).

Stability studies for the identification of critical process parameters for a pharmaceutical production of the Orf virus.

A promising new vaccine platform is based on the Orf virus, a viral vector of the genus Parapoxvirus, which is currently being tested in phase I clinical trials. The application as a vaccine platform mandates a well-characterised, robust, and efficient production process. To identify critical process parameters in the production process affecting the virus' infectivity, the Orf virus was subjected to forced degradation studies, including thermal, pH, chemical, and mechanical stress conditions. The tests indicated a robust virus infectivity within a pH range of 5-7.4 and in the presence of the tested buffering substances (TRIS, HEPES, PBS). The ionic strength up to 0.5 M had no influence on the Orf virus' infectivity stability for NaCl and MgCl2, while NH4Cl destabilized significantly. Furthermore, short-term thermal stress of 2d up to 37 °C and repeated freeze-thaw cycles (20cycles) did not affect the virus' infectivity. The addition of recombinant human serum albumin was found to reduce virus inactivation. Last, the Orf virus showed a low shear sensitivity induced by peristaltic pumps and mixing, but was sensitive to ultrasonication. The isoelectric point of the applied Orf virus genotype D1707-V was determined at pH3.5. The broad picture of the Orf virus' infectivity stability against environmental parameters is an important contribution for the identification of critical process parameters for the production process, and supports the development of a stable pharmaceutical formulation. The work is specifically relevant for enveloped (large DNA) viruses, like the Orf virus and like most vectored vaccine approaches.

A gradual transition from veridical to categorical representations along the visual hierarchy during working memory, but not perception.

The ability to stably maintain visual information over brief delays is central to cognitive functioning. One possible way to achieve robust working memory maintenance is by having multiple concurrent mnemonic representations across multiple cortical loci. For example, early visual cortex might contribute to storage by representing information in a "sensory-like" format, while intraparietal sulcus uses a format transformed away from sensory driven responses. As an explicit test of mnemonic code transformations along the visual hierarchy, we quantitatively modeled the progression of veridical-to-categorical orientation representations in human participants. Participants directly viewed, or held in mind, an oriented grating pattern, and the similarity between fMRI activation patterns for different orientations was calculated throughout retinotopic cortex. During direct perception, similarity was clustered around cardinal orientations, while during working memory the obliques were represented more similarly. We modeled these similarity patterns based on the known distribution of orientation information in the natural world: The "veridical" model uses an efficient coding framework to capture hypothesized representations during visual perception. The "categorical" model assumes that different "psychological distances" between orientations result in orientation categorization relative to cardinal axes. During direct perception, the veridical model explained the data well in early visual areas, while the categorical model did worse. During working memory, the veridical model only explained some of the data, while the categorical model gradually gained explanatory power for increasingly anterior retinotopic regions. These findings suggest that directly viewed images are represented veridically, but once visual information is no longer tethered to the sensory world, there is a gradual progression to more categorical mnemonic formats along the visual hierarchy.

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography.

Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set-up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3-5 µm pores in diameter instead of 1 µm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing.

Bioequivalence of Intravenous Alteplase from Two Different Manufacturing Processes in Healthy Male Volunteers: Results from a Two-Stage, Adaptive-Design Study.

OBJECTIVE: Alteplase is a recombinant tissue plasminogen activator used for thrombolytic treatment in several indications and is currently approved in Europe under the brand name Actilyse®. The current manufacturing process for alteplase was recently modified to meet increasing global demands. The aim of this randomized, open-label, adaptive two-stage design, two-way crossover study was to establish bioequivalence of alteplase derived from the two manufacturing processes (modified versus current). METHODS: The two alteplase formulations (modified and current, 0.2 mg/kg body weight) were compared in healthy male volunteers after intravenous infusion over a period of 30 min. The trial was put on hold after treatment of 12 subjects (Part A) and restarted as Part B (n = 18) with design adaptations, including a heparin bolus. RESULTS: Pharmacokinetic parameters of alteplase were determined from plasma concentration-time profiles. The pharmacokinetic parameters tested (AUC0-tz, Cmax, and AUC0-∞) for alteplase after single intravenous infusion demonstrated no differences between alteplase obtained from the modified and current processes. An analysis of variance (ANOVA) model was applied to test for bioequivalence. The geometric means ratio and the respective 92.83% confidence intervals (CIs) for all primary and secondary pharmacokinetic endpoints were well within the prespecified equivalence boundaries of 80-125%. The CIs also included unity, suggesting no statistically significant differences between the two treatments. CONCLUSIONS: The results show that alteplase exposure was virtually identical for the formulations tested, and statistical evaluation demonstrated bioequivalence of the formulations. Both formulations of alteplase were well tolerated by the subjects at the single intravenous doses in the trial. TRIAL REGISTRATION: Trial registration number: NCT04419493, 2019-004932-40 (EudraCT Number).

Evaluating Novel Quantification Methods for Infectious Baculoviruses.

Accurate and rapid quantification of (infectious) virus titers is of paramount importance in the manufacture of viral vectors and vaccines. Reliable quantification data allow efficient process development at a laboratory scale and thorough process monitoring in later production. However, current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring. Accordingly, flow cytometry and quantitative polymerase chain reaction have attracted increasing interest in recent years, offering various advantages for rapid quantification. Here, we compared different approaches for the assessment of infectious viruses, using a model baculovirus. Firstly, infectivity was estimated by the quantification of viral nucleic acids in infected cells, and secondly, different flow cytometric approaches were investigated regarding analysis times and calibration ranges. The flow cytometry technique included a quantification based on post-infection fluorophore expression and labeling of a viral surface protein using fluorescent antibodies. Additionally, the possibility of viral (m)RNA labeling in infected cells was investigated as a proof of concept. The results confirmed that infectivity assessment based on qPCR is not trivial and requires sophisticated method optimization, whereas staining of viral surface proteins is a fast and feasible approach for enveloped viruses. Finally, labeling of viral (m)RNA in infected cells appears to be a promising opportunity but will require further research.

The diverse role of heparan sulfate and other GAGs in SARS-CoV-2 infections and therapeutics.

In December 2019, the global coronavirus disease 2019 (COVID-19) pandemic began in Wuhan, China. COVID-19 is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infects host cells primarily through the angiotensin-converting enzyme 2 (ACE2) receptor. In addition to ACE2, several studies have shown the importance of heparan sulfate (HS) on the host cell surface as a co-receptor for SARS-CoV-2-binding. This insight has driven research into antiviral therapies, aimed at inhibiting the HS co-receptor-binding, e.g., by glycosaminoglycans (GAGs), a family of sulfated polysaccharides that includes HS. Several GAGs, such as heparin (a highly sulfated analog of HS), are used to treat various health indications, including COVID-19. This review is focused on current research on the involvement of HS in SARS-CoV-2 infection, implications of viral mutations, as well as the use of GAGs and other sulfated polysaccharides as antiviral agents.