Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16^INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.     

Structural Brain Changes in Chronic Pain Reflect Probably Neither Damage Nor Atrophy

Chronic pain appears to be associated with brain gray matter reduction in areas ascribable to the transmission of pain. The morphological processes underlying these structural changes, probably following functional reorganisation and central plasticity in the brain, remain unclear. The pain in hip osteoarthritis is one of the few chronic pain syndromes which are principally curable. We investigated 20 patients with chronic pain due to unilateral coxarthrosis (mean age 63.25±9.46 (SD) years, 10 female) before hip joint endoprosthetic surgery (pain state) and monitored brain structural changes up to 1 year after surgery: 6–8 weeks, 12–18 weeks and 10–14 month when completely pain free. Patients with chronic pain due to unilateral coxarthrosis had significantly less gray matter compared to controls in the anterior cingulate cortex (ACC), insular cortex and operculum, dorsolateral prefrontal cortex (DLPFC) and orbitofrontal cortex. These regions function as multi-integrative structures during the experience and the anticipation of pain. When the patients were pain free after recovery from endoprosthetic surgery, a gray matter increase in nearly the same areas was found. We also found a progressive increase of brain gray matter in the premotor cortex and the supplementary motor area (SMA). We conclude that gray matter abnormalities in chronic pain are not the cause, but secondary to the disease and are at least in part due to changes in motor function and bodily integration.     

A spontaneous point mutation in the Rubisco large subunit gene impairing holoenzyme assembly renders the IVβ plastome mutant of Oenothera extremely light‐ and chilling sensitive

A spontaneously occurring chloroplast genome (plastome) mutant of Oenothera , IVβ, was identified as a single point mutation in the Rubisco large subunit gene (G337 → C), leading to an V113L exchange, which topologically occurs at the interface of two adjacent large subunits (LSU). The minor sterical hindrance of dimer formation by this amino acid exchange strongly impairs holoenzyme assembly, leading to an accumulation of a processing precursor of the holoenzyme, the B‐complex, consisting of one LSU and 14 units of chaperonine 60 (cpn60). It is associated with very low holoenzyme concentrations in the mutant tissue, but does not affect the kinetic properties of the enzyme once assembled. When grown under moderate or low light, leaf tissue containing the plastome mutant showed decreased Chl contents and Chl a / b ratios, increased relative carotenoid contents and violaxanthin deepoxidation activity, but very low CO₂ fixation and O₂ evolution rates and was very sensitive to photoinhibition. The light dependence of chlorophyll fluorescence quenching components at low temperature resembled an extremely chilling sensitive Oenothera genotype as compared to the wild‐type. The IVβ mutant thus behaves similarly to the Rubisco SSU antisense plants analysed by Stitt and co‐workers (summarised by Stitt and Schulze 1994) and gives an example of the possible influence of plastome mutations on the sensitivity of the photosynthetic apparatus to excess light by modifying the capacity of the Calvin cycle.     

EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGFα showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGFα also play a role in mammary gland physiology during pregnancy by final developing and maintanance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA₂-arachidonic acid signalling cascade.     

A Cilia Independent Role of Ift88/Polaris during Cell Migration

Ift88 is a central component of the intraflagellar transport (Ift) complex B, essential for the building of cilia and flagella from single cell organisms to mammals. Loss of Ift88 results in the absence of cilia and causes left-right asymmetry defects, disordered Hedgehog signaling, and polycystic kidney disease, all of which are explained by aberrant ciliary function. In addition, a number of extraciliary functions of Ift88 have been described that affect the cell-cycle, mitosis, and targeting of the T-cell receptor to the immunological synapse. Similarly, another essential ciliary molecule, the kinesin-2 subunit Kif3a, which transports Ift-B in the cilium, affects microtubule (MT) dynamics at the leading edge of migrating cells independently of cilia. We now show that loss of Ift88 impairs cell migration irrespective of cilia. Ift88 is required for the polarization of migrating MDCK cells, and Ift88 depleted cells have fewer MTs at the leading edge. Neither MT dynamics nor MT nucleation are dependent on Ift88. Our findings dissociate the function of Ift88 from Kif3a outside the cilium and suggest a novel extraciliary function for Ift88. Future studies need to address what unifying mechanism underlies the different extraciliary functions of Ift88.     

The genus Alphitobius Stephens (Coleoptera,Tenebrionidae, Alphitobiini) in Africa and adjacent islands

All species of the genus Alphitobius Stephens, 1829 (Alphitobiini Reitter, 1917, subfamily Tenebrioninae Latreille, 1802) from Africa and adjacent islands are revised. New species: Alphitobius capitaneus sp. n. from Kenya. New synonyms: Cryptops ulomoides Solier, 1851, syn. n. of Alphitobius diaperinus (Panzer, 1796); Alphitobius rufus Ardoin, 1976, syn. n. of Alphitobius hobohmi Koch, 1953); Peltoides (Micropeltoides) crypticoides Pic, 1916, syn. n. of Peltoides (Micropeltoides) opacus (Gerstaecker, 1871), comb. n. Homonym: Alphitobius ulomoides Koch, 1953 = Alphitobius arnoldi nom. n. New combinations from Alphitobius: Ulomoides basilewskyi (Ardoin, 1969), comb. n.; Peltoides (Micropeltoides) opacus (Gerstaecker, 1871), comb. n. Figures of all examined species are added and a species key is compiled.