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Genome editing of hematopoietic stem and progenitor cells is being developed for the treatment of several inherited disorders of the hematopoietic system. The adaptation of CRISPR-Cas9-based technologies to make precise changes to the genome, and developments in altering the specificity and efficiency, and improving the delivery of nucleases to target cells have led to several breakthroughs. Many clinical trials are ongoing, and several pre-clinical models have been reported that would allow these genetic therapies to one day offer a potential cure to patients with diseases where limited options currently exist. However, there remain several challenges with respect to establishing safety, expanding accessibility and improving the manufacturing processes of these therapeutic products. This review focuses on some of the recent advances in the field of genome editing of hematopoietic stem and progenitor cells and illustrates the ongoing challenges.

To understand the position of a pacing lead in the right ventricle and to correctly interpret fluoroscopy and intracardiac signals, good anatomical knowledge is required. The right ventricle can be separated into an inlet, an outlet, and an apical compartment. The inlet and outlet are separated by the septomarginal trabeculae, while the apex is situated below the moderator band. A lead position in the right ventricular apex is less desirable, last but not least due to the thin myocardial wall. Many leads supposed to be implanted in the apex are in fact fixed rather within the trabeculae in the inlet, which are sometimes difficult to pass. In the right ventricular outflow tract (RVOT), the free wall is easier to reach than the septal due to the fact that the RVOT wraps around the septum. A mid-septal position close to the moderator band is relatively simple to achieve and due to the vicinity of the right bundle branch may produce a narrower paced QRS complex. Special and detailed knowledge is necessary for His bundle and left bundle branch pacing.

On-target toxicity to normal cells is a major safety concern with targeted immune and gene therapies. Here, we developed a base editing (BE) approach exploiting a naturally occurring CD33 single nucleotide polymorphism leading to removal of full-length CD33 surface expression on edited cells. CD33 editing in human and nonhuman primate (NHP) hematopoietic stem and progenitor cells (HSPCs) protects from CD33-targeted therapeutics without affecting normal hematopoiesisin vivo, thus demonstrating potential for novel immunotherapies with reduced off-leukemia toxicity. For broader applications to gene therapies, we demonstrated highly efficient (>70%) multiplexed adenine base editing of the CD33 and gamma globin genes, resulting in long-term persistence of dual gene-edited cells with HbF reactivation in NHPs.In vitro, dual gene-edited cells could be enriched via treatment with the CD33 antibody-drug conjugate, gemtuzumab ozogamicin (GO). Together, our results highlight the potential of adenine base editors for improved immune and gene therapies.Graphical abstract

Hot embossing is a cost-effective and flexible fabrication technology with high replication accuracy for feature sizes as small as 50 nm. Here we develop a reinforced polydimethylsiloxane (PDMS) mold for hot embossing of cyclic olefin polymer (COP) sheets in the fabrication of microfluidic chips and demonstrate the method by fabricating chips for automated sample digitization in digital nucleic acid assays. The PDMS is hardened by adding an investment powder as a dopant and is constrained with an aluminum frame to prevent lateral expansion during hot pressing. The reinforced PDMS mold demonstrated excellent performance in hot embossing (180 °C, 103 kPa, 5 min) for micropatterning COP sheets, with highly reproducible features as small as 10 μm (width of draining channel). In contrast, the microscale features were inconsistent and distorted when omitting either the investment powder or frame from the PDMS mold. COP chips were assembled by thermally bonding patterned and unpatterned COP sheets. We tested the performance of the COP chip for automated sample digitization in a digital LAMP assay used to quantify human papillomavirus-18 (HPV-18) DNA. A mixture of nucleic acid amplification reagents was loaded into the main channel of the chip using a syringe pump, then the solution was spontaneously partitioned into chambers (∼0.6 nL), which were then isolated by flowing oil through the chip. The digital LAMP assay produced accurately absolute quantitation of DNA at concentrations ranging from 10 to 1000 copies per μL. The strategy presented here provides a simple, low-cost method to prepare molds for hot embossing, which facilitates rapid validation of microfluidic designs.

Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multi-lineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/- HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome-editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.

Autorzy: Abe K., Adrich P., Aihara H., Akutsu, R., Alekseev I., Ali A. , Ameli F., Anghel I., Anthony L. H. V., Antonova M. , Araya A., Asaoka Y., Ashida Y., Aushev V., Ballester F., Bandac I., Barbi M., Barker G. J., Barr G., Batkiewicz-Kwasniak M., Bellato M., Berardi V., Bergevin M., Bernard L., Bernardini E., Berns L., Bhadra S., Bian J., Blanchet A., Blaszczyk F. d. M., Blonde A., Boiano A., Bolognesi S., Bonavera L., Booth N., Borjabad S., Boschi, T., Bose D., Boyd S . B., Bozza C., Bravar A., Bravo-Berguño D., Bronner C., Brown L., Bubak Arkadiusz, Buchowicz A., Buizza Avanzini M., Cafagna F. S., Calabria N. F., Calvo-Mozota J. M., Cao S., Cartwright S.L., Carroll A., Catanesi M. G., Cebriàn, S., Chabera M., Chakraborty, S., Checchia C., Choi J.H., Choubey S., Cicerchia M., Coleman J., Collazuol G., Cook L., Cowan G., Cuen-Rochin, S., Danilov M., Díaz López G., De la Fuente E., de Perio P., De Rosa G., Dealtry T., Densham C. J., Dergacheva A., Deshmukh N., Devi M. M., Di Lodovico F., Di Meo, P., Di Palma I., Doyle T. A., Drakopoulou E., Drapier O., Dumarchez J., Dunne P., Dziewiecki M., Eklund L., El Hedri S., Ellis J., Emery S., Esmaili A., Esteve R., Evangelisti A., Feely M., Fedotov S., Feng J., Fernandez P., Fernández-Martinez E., Ferrario P., Ferrazzi,B., Feusels T., Finch A., Finley C., Fiorentini A., Fiorillo G., Fitton M., Frankiewicz K., Friend M., Fujii Y., Fukuda Y., Galinski G., Gao J., Garde C., Garfagnini A., Garode S., Gialanella L., Giganti C., Gomez-Cadenas J.J., Gonin M., González-Nuevo J., Gorin A., Gornea R., Gousy-Leblanc V. Gramegna F. Grassi M. Grella G. Guigue M. Gumplinger P. Hadley D.R. Harada M., Hartfiel B., Hartz M., Hassani S., Hastings N.C., Hayato Y., Hernando-Morata J.A., Herrero V., Hill J., Hiraide K., Hirota S., Holin A., Horiuchi S., Hoshina K., Hultqvist K., Iacob F., Ichikawa A.K., Idrissi Ibnsalih W., Iijima T., Ikeda M., Inomoto M., Inoue K., Insler J., Ioannisian A., Ishida T., Ishidoshiro K., Ishino H., Ishitsuka M., Ito H., Ito S., Itow Y., Iwamoto K., Izmaylov A., Izumi N., Izumiyama S., Jakkapu M., Jamieson B., Jang H.I., Jang J.S., Jenkins S.J., Jeon S.H., Jiang M., Jo H.S., Jonsson P., Joo K.K., Kajita T., Kakuno H., Kameda J., Kano Y., Kalaczynski P., Karlen D., Kasperek J., Kataoka Y., Kato A., Katori T., Kazarian N., Kearns E., Khabibullin M., Khotjantsev A., Kikawa T., Kekic M., Kim J.H., Kim J.Y., Kim S.B., Kim S.Y., King S., Kinoshita T., Kisiel Jan, Klekotko A., Kobayashi T., Koch L., Koga M., Koerich L., Kolev N., Konaka A., Kormos L.L., Koshio Y., Korzenev A., Kotsar Y., Kouzakov K.A., Kowalik K.L., Kravchuk L., Kryukov A.P., Kudenko Y., Kumita T., Kurjata R., Kutter T., Kuze M., Kwak K., La Commara M., Labarga L., Lagoda J., Lamers James J., Lamoureux M., Laveder M., Lavitola L., Lawe M., Learned J.G., Lee J., Leitner R., Lezaun V., Lim I.T., Lindner T., Litchfield R.P., Long K.R., Longhin A., Loverre P., Lu X., Ludovici L., Maekawa Y., Magaletti L., Magar K., Mahn K., Makida Y., Malek M., Malinský M., Marchi T., Maret L., Mariani C., Marinelli A., Martens K., Marti L., Martin J.F. Martin D., Marzec J., Matsubara T., Matsumoto R., Matsuno S., Matusiak M., Mazzucato E., McCarthy M., McCauley N., McElwee J., McGrew C., Mefodiev A., Medhi A., Mehta P., Mellet L., Menjo H., Mermod P., Metelko C., Mezzetto M., Migenda J., Migliozzi P., Mijakowski P., Miki S., Miller E.W., Minakata H., Minamino A., Mine S., Mineev O., Mitra A., Miura M., Moharana R., Mollo C.M., Mondal T., Mongelli M., Monrabal F., Moon D.H., Moon C.S., Mora F.J., Moriyama S., Mueller Th.A., Munteanu L., Murase K., Nagao Y., Nakadaira T., Nakagiri K., Nakahata M., Nakai S., Nakajima Y., Nakamura K., Nakamura KI., Nakamura H., Nakano Y., Nakaya T., Nakayama S., Nakayoshi K., Nascimento Machado L., Naseby C.E.R., Navarro-Garcia B., Needham M., Nicholls T., Niewczas K., Nishimura Y., Noah E., Nova F., Nugent J.C., Nunokawa H., Obrebski W., Ochoa-Ricoux J.P., O’Connor E., Ogawa N., Ogitsu T., Ohta K., Okamoto K., O’Keeffe H.M., Okumura K., Onishchuk Y., Orozco-Luna F., Oshlianskyi A., Ospina N., Ostrowski M., O’Sullivan E., O’Sullivan L., Ovsiannikova T., Oyama Y., Ozaki H., Pac M.Y., Paganini P., Palladino V., Paolone V., Pari M., Parsa S., Pasternak J., Pastore C., Pastuszak G., Patel D.A., Pavin M., Payne D., Peña-Garay C., Pidcott C., Pinzon Guerra E., Playfer S., Pointon B.W., Popov A., Popov B., Porwit Kamil, Posiadala-Zezula M., Poutissou J.M., Pozimski J., Pronost G., Prouse N.W., Przewlocki P., Quilain B., Quiroga A.A., Radicioni E., Radics B., Rajda P.J., Renner J., Rescigno M., Retiere F., Ricciardi G., Riccio C., Richards B., Rondio E., Rose H.J., Roskovec B., Roth S., Rott C., Rountree S.D., Rubbia A., Ruggeri A.C., Ruggles C., Russo S., Rychter A., Ryu D., Sakashita K., Samani S., Sánchez F., Sánchez M.L., Sanchez M.C., Sano S., Santos J.D., Santucci G., Sarmah P., Sashima I., Sato K., Scott M., Seiya Y., Sekiguchi T., Sekiya H., Seo J.W., Seo S.H., Sgalaberna D., Shaikhiev A., Shan Z., Shaykina A., Shimizu I., Shin C.D., Shinoki M., Shiozawa M., Sinnis G., Skrobova N., Skwarczynski K., Smy M.B., Sobczyk J., Sobel H.W., Soler F. J. P., Sonoda Y., Spina R., Spisso B., Spradlin B., Stankevich K.L., Stawarz L., Stellacci S.M., Stopa K., Studenikin A.I., Suárez Gómez S.L., Suganuma T., Suvorov S., Suwa Y., Suzuki A.T., Suzuki S.Y., Suzuki Y., Svirida D., Svoboda R., Taani M., Tada M., Takeda A., Takemoto Y., Takenaka A., Taketa A., Takeuchi Y., Takhistov V., Tanaka H., Tanaka H.A., Tanaka H.I., Tanaka M., Tashiro T., Thiesse M., Thompson L.F., Toledo J., Tomatani-Sánchez A.K., Tortone G., Tsui K.M., Tsukamoto T., Tzanov M., Uchida Y., Vagins M.R., Valder S., Valentino V., Vasseur G., Vijayvargi A., Vilela C., Vinning W. G. S., Vivolo D., Vladisavljevic T., Vogelaar R.B., Vyalkov M.M., Wachala T., Walker J., Wark D., Wascko M.O., Wendell R.A., Wilkes R.J., Wilking M.J., Wilson M.R., Wronka S., Xia J., Xie Z., Xin T., Yamaguchi Y., Yamamoto K., Yanagisawa C., Yano T., Yen S., Yershov N., Yeum D.N., Yokoyama M., Yonenaga M., Yoo J., Yu I., Yu M., Zakrzewski T., Zaldivar B., Zalipska J., Zaremba K., Zarnecki G., Ziembicki M., Zietara K., Zito M., Zsoldos S., Core-collapse supernovae are among the most magnificent events in the observable universe. They produce many of the chemical elements necessary for life to exist and their remnants-neutron stars and black holes-are interesting astrophysical objects in their own right. However, despite millennia of observations and almost a century of astrophysical study, the explosion mechanism of core-collapse supernovae is not yet well understood. Hyper-Kamiokande is a next-generation neutrino detector that will be able to observe the neutrino flux from the next galactic core-collapse supernova in unprecedented detail. We focus on the first 500 ms of the neutrino burst, corresponding to the accretion phase, and use a newly-developed, high-precision supernova event generator to simulate Hyper-Kamiokandeʼs response to five different supernova models. We show that Hyper-Kamiokande will be able to distinguish between these models with high accuracy for a supernova at a distance of up to 100 kpc. Once the next galactic supernova happens, this ability will be a powerful tool for guiding simulations toward a precise reproduction of the explosion mechanism observed in nature.

In this paper, a compact wearable antipodal Vivaldi antenna resonating at 3.5 GHz is proposed for 5G n77 and n78 bands. It is designed on a flexible polyester substrate with a dielectric constant (ɛ r ) of 2 and loss tangent (tan δ) of 0.005. The antenna parameters were optimized via parametric analyses using CST software with a size of 33 × 33 mm² (length × width). The antenna is evaluated in terms of reflection coefficient (S 11 ), gain, efficiency, radiation pattern and surface current density and its reflection coefficient is verified with measurement. This antenna attained a maximum simulated gain of 4.17 dBi and an efficiency of 98.18 % in the resonating band.

This work presents a patch antenna with defected ground structure (DGS) on flexible materials for wearable applications. It is fed a 50 Ω inset feed and designed for operation at 2.45 GHz on a thermoplastic polyurethane-polyester fabric substrate. The DGS are integrated to ensure a compact design and improved bandwidth, efficiency, and gain. Simulations indicated that the antenna bandwidth is enhanced from 28.4 MHz without DGS to 79.3 MHz. Besides that, its gain is also improved from 2.01 dBi without DGS to 3.62 dBi and efficiency from 30% to 74%. Both antenna designs are fabricated and measured and provide good results and performance with efficiency greater than 50%.

Wrong-Way Driving (WWD) crashes have higher fatality rate than other crashes. In contrast to much of the existing literature, which has focused on collision data available after a WWD crash...

Protein complexes are the fundamental units of many biological functions. Despite their many advantages, one major adverse impact of protein complexes is accumulations of unassembled subunits that may disrupt other processes or exert cytotoxic effects. Synthesis of excess subunits can be inhibited via negative feedback control or they can be degraded more efficiently than assembled subunits, with this latter being termed cooperative stability. Whereas controlled synthesis of complex subunits has been investigated extensively, how cooperative stability acts in complex formation remains largely unexplored. To fill this knowledge gap, we have built quantitative models of heteromeric complexes with or without cooperative stability and compared their behaviours in the presence of synthesis rate variations. A system displaying cooperative stability is robust against synthesis rate variations as it retains high dimer/monomer ratios across a broad range of parameter configurations. Moreover, cooperative stability can alleviate the constraint of limited supply of a given subunit and makes complex abundance more responsive to unilateral upregulation of another subunit. We also conducted an in silico experiment to comprehensively characterize and compare four types of circuits that incorporate combinations of negative feedback control and cooperative stability in terms of eight systems characteristics pertaining to optimality, robustness and controllability. Intriguingly, though individual circuits prevailed for distinct characteristics, the system with cooperative stability alone achieved the most balanced performance across all characteristics. Our study provides theoretical justification for the contribution of cooperative stability to natural biological systems and represents a guideline for designing synthetic complex formation systems with desirable characteristics.

The search for a dark photon holds considerable interest in the physics community. Such a force carrier would begin to illuminate the dark sector. Many experiments have searched for such a particle, but so far it has proven elusive. In recent years the concept of a low mass dark photon has gained popularity in the physics community. Of particular recent interest is the 8Be and 4He anomaly, which could be explained by a new fifth force carrier with a mass of 17 MeV/c 2. The proposed Darklight experiment would search for this potential low mass force carrier at ARIEL in the 10-20 MeV/c 2 e+e− invariant mass range. This proceeding will focus on the experimental design and physics case of the Darklight experiment.

SUMMARYGlutathione (GSH) is a small molecule thiol abundantly present in all eukaryotes with key roles in oxidative metabolism1. Mitochondria, as the major site of oxidative reactions, must maintain sufficient levels of GSH to perform protective and biosynthetic functions2. GSH is exclusively synthesized in the cytosol, yet the molecular machinery involved in mitochondrial GSH import remain elusive. Here, using organellar proteomics and metabolomics approaches, we identify SLC25A39, a mitochondrial membrane carrier of unknown function, to regulate GSH transport into mitochondria. SLC25A39 loss reduces mitochondrial GSH import and abundance without impacting whole cell GSH levels. Cells lacking both SLC25A39 and its paralog SLC25A40 exhibit defects in the activity and stability of ironsulfur cluster containing proteins. Moreover, mitochondrial GSH import is necessary for cell proliferation in vitro and red blood cell development in mice. Remarkably, the heterologous expression of an engineered bifunctional bacterial GSH biosynthetic enzyme (GshF) in mitochondria enabled mitochondrial GSH production and ameliorated the metabolic and proliferative defects caused by its depletion. Finally, GSH availability negatively regulates SLC25A39 protein abundance, coupling redox homeostasis to mitochondrial GSH import in mammalian cells. Our work identifies SLC25A39 as an essential and regulated component of the mitochondrial GSH import machinery.