A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database (http://www. hidras.unimi.it) to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.

WD40 repeat proteins similar to yeast MSI1 are conserved in animals and plants, in which they participate in complexes involved in chromatin metabolism. Although MSI1-like proteins are well characterised biochemically,their function in the development of multicellular eukaryotes is not well understood. We constructed Arabidopsis plants in which the AtMSI1 protein level was altered. Strong ectopic expression of AtMSI1 produced no visible altered phenotype, but reduction of AtMSI1 dramatically affected development. The primary shoot apical meristem was unable to develop organs after the transition to flowering. Flowers that developed on floral shoots from axillary meristems experienced a progressive loss of floral morphology,including a reduction in size of the petals and stamens and the development of carpel-like sepals. Ovule development was disrupted in all flowers, resulting in complete female sterility. Molecular analysis of the mutant plants revealed that AtMSI1 is required to maintain the correct temporal and organ-specific expression of homeotic genes, including AGAMOUS and APETALA2. In contrast, FAS1 and FAS2, which together with AtMSI1 form the chromatin assembly complex CAF-1, are not required for repression of these genes. Therefore, AtMSI1 has specific functions in addition to CAF-1-mediated chromatin assembly. Efficient formation of heterochromatin, but not methylation of centromeric DNA repeats, depends on AtMSI1 presence demonstrating a key role of AtMSI1 in maintenance of chromatin structure.

Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins (∼15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naïve library of 1012 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).

For all known major apple scab resistance genes except Vr, molecular markers have been published. However, the precise position of some of these genes, in the apple genome, remains to be identified. Knowledge about the relative position of apple scab resistance genes is necessary to preliminarily evaluate the probability of success of their pyramidization. Pyramidization of different resistance genes into the same genotype is a reliable way to create cultivars with durable apple scab resistance. Applying the genome scanning approach (GSA), we identified the linkage group of the scab resistance gene Vm, derived from Malus micromalus, and we found a new molecular marker tightly associated with the gene. The simple sequence repeat Hi07h02, previously mapped on linkage group 17, cosegregates with the Vm gene (no recombinants in the 95 plants tested). The already published sequence-characterized amplified region Vm marker OPB12(687) was found to be linked at about 5 cM from the resistance gene and, therefore, this marker also maps on linkage group 17 of apple. This is the first report of the discovery of a major apple scab resistance gene on linkage group 17. The advantages of using GSA for the identification of molecular markers for qualitative traits are discussed.

Microencapsulation gains increasing importance for processing of bacterial libraries and especially in high-throughput (HT) environments where [10 6 samples per day are studied. As a rule, a one-to-one relationship between an individual cell and analytical results is of key importance. Ideally, each microcarrier would therefore contain exactly one cell or colony. However, synthesis of larger numbers of capsules containing exactly one cell is not feasible as cells are randomly distributed during carrier-production. The dilemma is that high dilution conditions will yield a satisfactory degree of monoclonality, but also a very large fraction of empty compartments, whereas distribution under low dilution generates unacceptable numbers of polyclonal compartments for whose removal no satisfactory technologies exist. Hydrogel carriers with a volume of 35 nL were used as growth compartments for individual microbial colonies. E. coli cells expressing green fluorescent protein (GFP) were encapsulated at low dilution thereby intentionally producing a considerable amount of polyclonal microcarrieres. Empty and polyclonal microcarriers were then removed from the desired monoclonal fraction by a COPAS Plus particle analyzer. The results were compared with model predictions in order to investigate possible limitations in the analysis and sorting of monoclonal microcarriers by COPAS. Fluorescent E. coli cells (GFP) distributed randomly throughout the microcarrier population. Cells were successfully propagated to colonies in the microcarriers and enriched to 95% monoclonality by a COAPS sorter. Enrichment-efficiency was found to mainly depend on the colony diameter. With increasing colony size two contrary effects were observed: First, improved sorting efficiency due to increased fluorescence intensity and therefore higher detection efficiency, and second, deterioration of sorting efficiency due to occlusion occurring in polyclonal carriers. The combination of microencapsulation under low dilution conditions followed by HT sorting procedures is an efficient way for isolating larger amount of monoclonal carriers from bacterial libraries while concomitantly keeping the amounts of empty carriers at a moderate level. ' International Society for Advancement of CytometryKey terms microencapsulation; monoclonality; microcarrier; in vitro compartmentalization (IVC); fluorescent E. coli; COPAS plus particle analyzer and sorter; ultrahigh throughput screening THE enormous complexity of living systems makes high-throughput (HT) experimenting and analyses an indispensable tool for research and development in the lifeand biosciences. Sample numbers exceeding 10 6 per day (1-4) require new technologies for sample miniaturization and highly parallelized processing (5). Naturally, most experimental strategies rely on formation of spatially separated monoclonal cell assemblies at the beginning. If microbial species are studied, an approach that is frequently employed for generating larger numbers of monoseptic compartments is to first ...

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has so far infected more than 100 million people globally, and continues to undergo genomic evolution. Emerging SARS-CoV-2 variants show increased infectivity and may lead to resistance against immune responses of previously immunized individuals or existing therapeutics, especially antibody-based therapies. Several monoclonal antibody therapeutics authorized for emergency use or in development start to lose potency against various SARS-CoV-2 variants. Cocktails of two different monoclonal antibodies constitute a promising approach to protect against such variants as long as both antibodies are potent, but come with increased development complexity and therefore cost. As an alternative, we developed two multi-specific DARPin® therapeutics, each combining three independent DARPin® domains binding the SARS-CoV-2 spike protein in one molecule, to potently neutralize the virus and overcome virus escape. Here, we show in a panel of in vitro studies that both multi-specific DARPin® therapeutics, ensovibep (MP0420) and MP0423, are highly potent against the new circulating SARS-CoV-2 variants B.1.1.7 (UK variant) and B.1.351 (South African variant) and the most frequent emerging mutations in the spike protein. Additionally, viral passaging experiments show potent protection by ensovibep and MP0423 against development of escape mutations. Furthermore, we demonstrate that the cooperative binding of the individual modules in a multi-specific DARPin® antiviral is key for potent virus inhibition and protection from escape variants. These results, combined with the relatively small size and high production yields of DARPin® molecules, suggests ensovibep and MP0423 as superior alternatives to monoclonal antibody cocktails for global supply and demonstrate the strength of the DARPin® platform for achieving potent and lasting virus inhibition for SARS-CoV-2 and possibly other viruses.

We introduce a technology for the rapid identification and sequencing of conserved DNA elements employing a novel suspension array based on nanoliter (nl)-reactors made from alginate. The reactors have a volume of 35 nl and serve as reaction compartments during monoseptic growth of microbial library clones, colony lysis, thermocycling and screening for sequence motifs via semi-quantitative fluorescence analyses. nl-Reactors were kept in suspension during all high-throughput steps which allowed performing the protocol in a highly space-effective fashion and at negligible expenses of consumables and reagents. As a first application, 11 high-quality microsatellites for polymorphism studies in cassava were isolated and sequenced out of a library of 20 000 clones in 2 days. The technology is widely scalable and we envision that throughputs for nl-reactor based screenings can be increased up to 100 000 and more samples per day thereby efficiently complementing protocols based on established deep-sequencing technologies.