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Cellular organelles and biological macromolecules such as proteins play a fundamental role in almost all life sciences. Structural biology studies the molecular structure of these particles to gain information about their morphology and function. A quickly evolving technique in this field is cryo electron-tomography (cryo-ET). Its big advantage is the ability to determine the three dimensional structure of particles in their native environment. A powerful method for image analysis in cryo-ET is subtomogram averaging (STA), where repetitions of the same particle of interest within a tomogram are computationally extracted, aligned to a common reference and averaged. This procedure significantly increases the signal of the underlying structure. A main bottleneck in cryo-ET and STA is the low throughput in sample preparation, data acquisition and image processing. With the goal to overcome this challenge, we developed computational methods to automate and streamline key steps in image processing. Two projects were designed for this purpose: In the first project, we streamlined the complete processing pipeline of STA and minimized the manual user interaction. In the second project we developed a new algorithm to fully automate the alignment of tomographic tilt series. We further developed a range of specialized tools for STA. We applied them on biological samples in combination with our established methods for increased throughput. Thereby we determined the architecture of protein coats involved in membrane trafficking and shed light on the underlying mechanics. With our new methods we successfully reduced processing times for STA, answered biological questions and laid the foundation for further developments.

Summary Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41+ cells gradually gave rise to CD45+ cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting., Graphical Abstract, Highlights • iPSCs from oc/oc mice bearing Tcirg1 gene mutation were generated for the first time • A BAC-based approach corrects the Tcirg1 gene mutation • iPSCs differentiate similarly to physiologic fetal hematopoiesis • The osteopetrotic phenotype in osteoclasts from BAC-corrected iPSCs was rescued, In this article, Villa and colleagues present a multistep strategy by which iPSCs are generated from osteopetrotic mice, genetically corrected by homologous recombination using a BAC carrying the entire Tcirg1 gene locus, and differentiated toward hematopoietic early progenitors able to give rise to functional osteoclasts, rescuing the defective cellular phenotype.

Polymer-fullerene or polymer-polymer based bulk heterojunction (BHJ) solar cells can be fabricated by using low-cost manufacturing methods. However, because of the low mobility of organic materials, there is a competition between separation and recombination of the photogenerated carriers within the thin BHJ film. Thus, there is a need to develop strategies to increase light harvesting in the films without increasing the film thickness. Nanoparticles (NPs) have been receiving a lot of interest for exhibiting interesting optical, electrical, and magnetic properties. These novel properties can be exploited in nanotechnology by forming compact and ordered architectures of nanoparticles within of BHJ film. The addition of metal NPs in the BHJ films could ensure a greater absorption and an enhanced photogeneration of mobile carriers [1]. In this work, we present a study of the effect of thiol-capped Au nanoparticles (AuNPs), within an organic device, by controlling on the nanoscale the position of the nanoparticles in the different interfaces of the device. Au-NPs have been obtained by laser ablation in liquid solution (LASiS) [2] and have been functionalized both with 2-naphthalenethiol and alkanethiol having different length. Such particles in combination with poly(3-hexylthiophene) (P3HT), [6,6]-phenyl C61 butyric acid methyl ester (PCBM) and Poly[(9,9-di-n-octylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazol-4,8-diyl)] (F8BT) have been used to fabricate two different BHJ (P3HT:PCBM and P3HT:F8BT) with three components thin films. In particular we have realized planar and bulk heterojunction thin films by the Langmuir-Blodgett (LB) apparatus, which offers a simple method of producing ultra-thin films with fine control over thickness. We prepared at first planar heterojunction (PHJ) structures consisting of layers of P3HT, Au-NPs and PCBM or F8BT by the horizontal lifting (Langmuir-Schaefer LS) technique. Then we induced a transition from PHJ to BHJ by thermal annealing to mix the layers. To study the effect of the position of the nanoparticles, different structures have been realized, which differ in the position of the Au-NPs layer, i.e. at substrate/donor or donor/acceptor interfaces as well as at the top of the acceptor layer. These structures and their components have been studied by microscopy and spectroscopy surface tools and by electrochemistry to investigate the energy levels. I/V curves and importantly fluorescence quenching analysis showed that 2-naphthalenethiol-capped Au-NPs film incorporated at the P3HT/PCBM interface ensures a more efficient charge transfer if compared to the same heterojunction without AuNPs. The beneficial effect of nanoparticles seems to be lost after the transition from PHJ to BHJ, where there is any control over the particles location. Other important advantages on the efficiency of these three components devices are discussed.

We developed a clinically applicable gene transfer procedure into mobilized peripheral blood (MPB) CD34(+) hematopoietic progenitor cells, based on single viral exposure and selection of engineered cells. CD34(+) cells were transduced with a retroviral vector carrying the truncated form of the nerve growth factor receptor (DNGFR) marker gene, and immunoselected for DNGFR expression. Optimal time and procedure for viral exposure, length of culture, and transgene expression of MPB CD34(+) cells were determined using in vitro assays. The multipotent capacity of MPB CD34(+)-transduced cells was demonstrated in the SCID-hu bone/liver/thymus mouse model. Transduced DeltaNGFR(+) cells retained 50% of long-term culture-colony forming cells (LTC-CFC) compared to unmanipulated CD34(+) cells. In SCID-hu mice, 52% of CD45(+) cells, 27% of CD34(+) cells, 49% of B cells, and more than 50% of T cells were derived from transplanted CD34(+)/DeltaNGFR(+) cells. Furthermore, transplantation of purified transduced cells greatly reduced the competition with untransduced progenitors occurring in unselected grafts. These data demonstrate that MPB CD34(+) cells, transduced with a single viral exposure and selected by transgene expression, retain multilineage reconstitution capacity and remarkable transgene expression.

To the Editor: Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by thrombocytopenia, eczema, and immunodeficiency. Up to 70% of patients with WAS present with at least 1 autoimmune or autoinflammatory episode, and many of them suffer from recurrent or multiple events.1, 2, 3 IL-1 new-generation blockers have been used in patients exhibiting clinical symptoms compatible with an autoinflammatory condition,4 but have not been reported in WAS. Here, we describe a patient with WAS with a peculiar large genomic inversion presenting with multiple manifestations of immune dysregulation, in whom autoinflammatory manifestations improved after the use of anakinra (IL-1 receptor antagonist, Kineret). A 11.6-year-old boy was referred to our center for suspected immunodeficiency. The patient presented with a history of microthrombocytopenia since birth and eczema in the first years of life, suggestive of WAS. Analysis of WAS protein (WASp) expression was reported abnormal, but Sanger sequencing on DNA did not reveal mutations. From 1.5 years of age he underwent recurrent episodes of postinfectious vasculitis of the lower limbs and arthritis. At 7.5 years, he presented with a bilateral pneumonia that triggered Schonlein-Henoch purpura with fever and arthritis, managed with oral steroids. Subsequently, a nephritic-nephrotic syndrome was treated with antihypertensive treatment and high-dose corticosteroids (CCS), with partial response. Cyclosporin A (CyA) and CCS led to remission of renal disease, which relapsed after CyA was stopped. Intravenous high-dose CCS and anti-CD20 mAb did not lead to substantial improvement. CyA and low-dose prednisone were restarted with partial benefit. However, the patient experienced varicella zoster reactivation on his half-right-face, with sequelae to the right eye (anterior and posterior uveitis with acute retinitis) requiring a vitrectomy, and severe impairment of visual function. An anterior uveitis at the left eye was treated with steroids. At the age of 9.8 years, he developed clinical and histological features of pancolitic Crohn disease, managed with an increase in CCS, as well as arthritis and histologically confirmed vasculitis and eventually pyoderma gangrenosum (PG) on the hips, buttocks, and upper and lower limbs. Crohn disease was not responsive to infliximab, thalidomide, cyclophosphamide, or high-dose intravenous steroids, while adalimumab (Humira) resulted in an initial benefit (see Table E1 in this article's Online Repository at www.jacionline.org). The patient presented with fistulas and perianal abscesses when he was 10.7 years old and he underwent several fistulectomies and removal of granulation tissue in the perianal area by “cone-like technique.” For the poor control of the enterocolitis, a subtotal colectomy with terminal ileostomy was performed at age 11 years. When the patient was referred to our center, he was on adalimumab and low-dose CCS with a good control of bowel disease, but still showed severe manifestations of PG on the upper limbs and in the perianal area (Fig 1, A; see Table E2 in this article's Online Repository at www.jacionline.org). His parents signed informed consent for research investigations (protocol Tiget06). Fig 1 Skin lesions and biochemical markers in a patient with WAS with autoinflammatory manifestations. A, Patient at the time of WAS diagnosis. B, Patient after 3 months of treatment with MTX. C, Patient after 5 months of treatment with anakinra. ... Because of the strong suspicion of WAS, whole-genome sequencing was performed and an inversion of 6kb spanning from the promoter to the intronic region between exons 7 and 8 was detected (see Fig E1 in this article's Online Repository at www.jacionline.org). Specific primers in this region identified the precise breaking points (see Tables E3 and ​andE4E4 in this article's Online Repository at www.jacionline.org; Fig 2, B). The rearranged allele was present in the patient and his mother, whereas the patient's aunt was unaffected (data not shown and Fig 2, A-C). Fig E1 Graphical representation of WGS results. WAS gene and coverage are indicated. Primers R1 and R2 for Illumina sequencing that pair correctly are represented in gray. The red lines in the patient indicate the pairing in the region spanning the inversion ... Fig 2 Identification of inversion in the WAS gene. A, Pedigree of the family. Proband is indicated by arrow. B, Graphical representation of predicted effects of inversion in the WAS gene. Primer design in the sites of inversion. C, DNA amplification with primers ... RNA analyses showed an aberrant transcript produced from the inverted region (Fig 2, D). WASp expression, analyzed by flow cytometry (see Fig E2, A, in this article's Online Repository at www.jacionline.org), was deeply reduced in peripheral blood T-, B-, and natural killer lymphocytes and monocytes (data not shown) while it was undetectable by Western blot performed with an antibody recognizing the N-terminal portion of WASp including exons 7 and 8 (Fig E2, B). WASp expression was restored in the patient's T-cell line transduced with a lentiviral vector expressing WASp under the control of the autologous 1.6-kb long promoter5 (Fig E2, C). Fig E2 WASp expression. A, Flow cytometry characterization of WASp expression in patient and HC lymphocytes. Percentage of WASp+ cells is reported on histograms. Detection of WASp was performed after permeabilization (Cytofix/Cytoperm kit; BD Biosciences, ... The start of low-dose methotrexate (Reumaflex) and the increase in prednisone led to a moderate improvement in the PG after 3 months (Fig 1, B), but shortly after the patient underwent a reactivation of vasculitis and arthritis with systemic inflammation that was not controlled by multiple immunosuppressive and anti-inflammatory drugs. On the basis of the reported efficacy of IL-1 blockers in the treatment of autoinflammatory manifestations and of PG,4 anakinra was started as an off-label drug titrating the dose from 1 up to 3 mg/kg/day. This led to a resolution of vasculitis and arthritis and to a decrease in the inflammation indexes within few days (Fig 1, D) with dramatic improvement in the PG skin lesions during the following 5 months (Fig 1, C). The patient was enrolled in a gene therapy trial based on autologous gene-corrected hematopoietic stem cells (clinicaltrials.gov #NCT01515462), mobilized with G-CSF and plerixafor. Treatment with anakinra was discontinued 48 hours before mobilization, but was soon restarted because of the increase in white blood cells and inflammation indexes with exacerbation of skin lesions, arthralgia, and hematuria, and led again to a rapid laboratory and clinical remission (data not shown). Notably, the use of anakinra allowed a successful mobilization with G-CSF without the occurrence of other autoinflammatory manifestations. To our knowledge, this is the first reported case of use of IL-1R blocker in a patient with WAS, with clinical benefit. This case is very emblematic for several reasons. Whole-genome sequencing complemented by specific breakpoint sequencing allowed the identification of the inversion with intact exon sequences, elucidating the previous normal genetic analysis. Complex genomic rearrangements involving inversions are generally noncanonical gene conversion events6 and could have occurred in an ancestor allele in the family through a de novo mutation occurring in the mother. Autoimmune and autoinflammatory manifestations in patients with WAS typically present early in life, are often refractory to therapy, and are associated with a worse clinical prognosis and an increased risk of developing a malignancy.3, 7 Our patient's autoinflammatory manifestations were resistant to several immunosuppressive drugs and the use of CyA was associated with a severe viral complication. Anakinra dramatically improved PG, vasculitis, and arthritis, showed a good safety profile, and allowed stabilization of the patient for definitive treatment. The response to anakinra suggests that the dysregulation of the innate immune system is involved in the genesis of autoinflammatory manifestations in patients with WAS and shows that IL-1 may serve in selected cases as a target for therapy, avoiding the use of other classes of immunosuppressors that can increase the risk for severe infections. It has been hypothesized that defects in chemotaxis and podosomes formation in WASp-deficient cells may favor the onset of autoinflammatory manifestations. In addition, a recent study in a patient with aggressive PG showed a critical role for proline-serine-threonine phosphatase interacting protein 1, which is involved in cytoskeletal regulatory functions through interaction with WASp, in the Pyogenic Arthritis, Pyoderma gangrenosum, and Acne syndrome.8 A greater understanding of the role of WASp in inflammation and of potential pathways that may be targeted therapeutically to modulate immunity in WAS is desirable to improve the management of the affected patients while waiting for definitive treatment by stem cell transplantation or gene therapy.

Gene transfer into autologous hematopoietic stem progenitor cells (HSPCs) has the potential to cure monogenic inherited disorders caused by an altered development and/or function of the blood system, such as immune deficiencies and red blood cell and platelet disorders. Gene-corrected HSPCs and their progeny can also be exploited as cell vehicles to deliver molecules into the circulation and tissues, including the central nervous system. In this review, we focus on the progress of clinical development of medicinal products based on HSPCs engineered and modified by integrating viral vectors for the treatment of monogenic blood disorders and metabolic diseases. Two products have reached the stage of market approval in the EU, and more are foreseen to be approved in the near future. Despite these achievements, several challenges remain for HSPC gene therapy (HSPC-GT) precluding a wider application of this type of gene therapy to a wider set of diseases while gene-editing approaches are entering the clinical arena. In this review, Tucci and colleagues describe the progress of current ex vivo gene therapy clinical trials on primary immunodeficiencies, hemoglobinopathies, and metabolic diseases, and they discuss the challenges that remain to be overcome.

Recombinant antibodies fragments in several new formats are routinely investigated and used in diagnostic and therapeutic applications as anti-cancers molecules. New antibody formats are generated to compensate the need for multispecificity and site-specific introduction of fluorescent dyes, cytotoxic payloads or for generating semisynthetic multimeric molecules. Fabs of trastuzumab bearing transglutaminase (MTG) reactive sites were generated by periplasmic expression in E. coli and purified. Multimeric Fabs were generated by either disulfide bridge formation or by using MTG-sensitive peptide linkers. Binding to receptor was assessed by ELISA and SPR methods. Internalization and growth inhibition assays were performed on BT-474 and SKBR3 Her2+ cells. Fabs were successfully produced and dimerized or trimerized using MTG and suitably designed peptide linkers. Site-specific derivatizations with fluorophores were similarly achieved. The monomeric, dimeric and trimeric variants bind the receptor with affinities similar or superior to the full antibody. Fab and Fab2 are rapidly internalized in Her2+ cells and exhibit growth inhibition abilities similar to the full antibody. Altogether, the data show that the recombinant Fabs can be produced in E. coli and converted into multimeric variants by MTG-based bioconjugation. Similar approaches are extendable to the introduction of cytotoxic payloads for the generation of novel Antibody Drug Conjugates.