Point-counterpoint: Should we be performing metagenomic next-generation sequencing for infectious disease diagnosis in the clinical laboratory?

With established applications of next-generation sequencing in inherited diseases and oncology, clinical laboratories are evaluating the use of metagenomics for identification of infectious agents directly from patient samples, to aid in the diagnosis of infections. Metagenomic next-generation sequencing for infectious diseases promises an unbiased approach to detection of microbes that does not depend on growth in culture or the targeting of specific pathogens. However, the issues of contamination, interpretation of results, selection of databases used for analysis, and prediction of antimicrobial susceptibilities from sequencing data remain challenges. In this Point-Counterpoint, Steve Miller and Charles Chiu discuss the pros of using direct metagenomic sequencing, while Kyle Rodino and Melissa Miller argue for the use of caution

The Gabor wave front set of compactly supported distributions

We show that the Gabor wave front set of a compactly supported distribution equals zero times the projection on the second variable of the classical wave front set

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

Background: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation. Methods: The capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the Nterminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD. Results: Regional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month), 91.3 ± 3.1 (2 months), and 89.6 ± 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8 (1 month), 78.9 ± 7.4 (2 months), and 81.2 ± 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 ± 0.3 (1 month), 2.1 ± 0.8 (2 months), and 2.1 ± 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 × 1012 vg/kg in mouse vs 2 × 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius – 63.8 ± 4.9%, lateral gastrocnemius – 66.0 ± 4.5%, EDL – 80.2 ± 3.1%, soleus – 86.4 ± 1.9%, TA – 72.2 ± 4.0%. Conclusion: These studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD

Persistent Expression of FLAG-tagged Micro dystrophin in Nonhuman Primates Following Intramuscular and Vascular Delivery

Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50–79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials

Orientia tsutsugamushi ankyrin repeat-containing protein family members are Type 1 secretion system substrates that traffic to the host cell endoplasmic reticulum

Scrub typhus is an understudied, potentially fatal infection that threatens one billion persons in the Asia-Pacific region. How the causative obligate intracellular bacterium, Orientia tsutsugamushi, facilitates its intracellular survival and pathogenesis is poorly understood. Many intracellular bacterial pathogens utilize the Type 1 (T1SS) or Type 4 secretion system (T4SS) to translocate ankyrin repeat-containing proteins (Anks) that traffic to distinct subcellular locations and modulate host cell processes. The O. tsutsugamushi genome encodes one of the largest known bacterial Ank repertoires plus T1SS and T4SS components. Whether these potential virulence factors are expressed during infection, how the Anks are potentially secreted, and to where they localize in the host cell are not known. We determined that O. tsutsugamushi transcriptionally expresses 20 unique ank genes as well as genes for both T1SS and T4SS during infection of mammalian host cells. Examination of the Anks’ C-termini revealed that the majority of them resemble T1SS substrates. Escherichia coli expressing a functional T1SS was able to secrete chimeric hemolysin proteins bearing the C-termini of 19 of 20 O. tsutsugamushi Anks in an HlyBD-dependent manner. Thus, O. tsutsugamushi Anks C-termini are T1SS-compatible. Conversely, Coxiella burnetii could not secrete heterologously expressed Anks in a T4SS-dependent manner. Analysis of the subcellular distribution patterns of 20 ectopically expressed Anks revealed that, while 6 remained cytosolic or trafficked to the nucleus, 14 localized to, and in some cases, altered the morphology of the endoplasmic reticulum. This study identifies O. tsutsugamushi Anks as T1SS substrates and indicates that many display a tropism for the host cell secretory pathway

Nuclearity of rapidly decreasing ultradifferentiable functions and time-frequency analysis

[EN] We use techniques from time-frequency analysis to show that the space S(omega )of rapidly decreasing omega-ultradifferentiable functions is nuclear for every weight function omega(t) = o(t) as t tends to infinity. Moreover, we prove that, for a sequence (M-p)(p) satisfying the classical condition (M1) of Komatsu, the space of Beurling type S-(M)p when defined with L-2 norms is nuclear exactly when condition (M2)' of Komatsu holds.We thank the reviewer very much for the careful reading of our manuscript and the comments to improve the paper. The first three authors were partially supported by the Project FFABR 2017 (MIUR), and by the Projects FIR 2018 and FAR 2018 (University of Ferrara). The first and third authors are members of the Gruppo Nazionale per l'Analisi Matematica, la Probabilita e le loro Applicazioni (GNAMPA) of the Istituto Nazionale di Alta Matematica (INdAM). The research of the second author was partially supported by the project MTM2016-76647-P and the grant BEST/2019/172 from Generalitat Valenciana. The fourth author is supported by FWF-project J 3948-N35.Boiti, C.; Jornet Casanova, D.; Oliaro, A.; Schindl, G. (2021). Nuclearity of rapidly decreasing ultradifferentiable functions and time-frequency analysis. Collectanea mathematica. 72(2):423-442. https://doi.org/10.1007/s13348-020-00296-0S423442722Asensio, V., Jornet, D.: Global pseudodifferential operators of infinite order in classes of ultradifferentiable functions. Rev. R. Acad. Cienc. Exactas Fís. Nat. Ser. A Mat. RACSAM 113(4), 3477–3512 (2019)Aubry, J.-M.: Ultrarapidly decreasing ultradifferentiable functions, Wigner distributions and density matrices. J. London Math. Soc. 2(78), 392–406 (2008)Björck, G.: Linear partial differential operators and generalized distributions. Ark. Mat. 6(21), 351–407 (1966)Boiti, C., Jornet, D., Oliaro, A.: Regularity of partial differential operators in ultradifferentiable spaces and Wigner type transforms. J. Math. Anal. Appl. 446, 920–944 (2017)Boiti, C., Jornet, D., Oliaro, A.: The Gabor wave front set in spaces of ultradifferentiable functions. Monatsh. Math. 188(2), 199–246 (2019)Boiti, C., Jornet, D., Oliaro, A.: About the nuclearity of $$\cal{S}_{(M_{p})}$$ and $$\cal{S}_{\omega }$$. In: Boggiatto, P., et al. (eds.) Advances in Microlocal and Time-Frequency Analysis. Applied and Numerical Harmonic Analysis, pp. 121–129. Birkhäuser, Cham (2020)Boiti, C., Jornet, D., Oliaro, A.: Real Paley-Wiener theorems in spaces of ultradifferentiable functions. J. Funct. Anal. 278(4), 108348 (2020)Bonet, J., Meise, R., Melikhov, S.N.: A comparison of two different ways to define classes of ultradifferentiable functions. Bull. Belg. Math. Soc. Simon Stevin 14(3), 425–444 (2007)Braun, R.W., Meise, R., Taylor, B.A.: Ultradifferentiable functions and Fourier analysis. Result. Math. 17, 206–237 (1990)Fernández, C., Galbis, A., Jornet, D.: Pseudodifferential operators on non-quasianalytic classes of Beurling type. Studia Math. 167(2), 99–131 (2005)Fernández, C., Galbis, A., Jornet, D.: Pseudodifferential operators of Beurling type and the wave front set. J. Math. Anal. Appl. 340(2), 1153–1170 (2008)Franken, U.: Weight functions for classes of ultradifferentiable functions. Results Math. 25, 50–53 (1994)Gröchenig, K.: Foundations of Time-Frequency Analysis. Birkhäuser, Boston (2001)Gröchenig, K., Leinert, M.: Wiener’s Lemma for twisted convolution and Gabor frames. J. Am. Math. Soc. 17(1), 1–18 (2004)Gröchenig, K., Zimmermann, G.: Spaces of Test Functions via the STFT. J. Funct. Spaces Appl. 2(1), 25–53 (2004)Heinrich, T., Meise, R.: A support theorem for quasianalytic functionals. Math. Nachr. 280(4), 364–387 (2007)Hörmander, L.: Notions of Convexity. Progress in Mathematics, vol. 127. Birkhäuser, Boston (1994)Janssen, A.J.E.M.: Duality and Biorthogonality for Weyl-Heisenberg Frames. J. Fourier Anal. Appl. 1(4), 403–436 (1995)Komatsu, H.: Ultradistributions I. Structure theorems and a characterization. J. Fac. Sci. Univ. Tokyo Sect IA Math. 20, 25–105 (1973)Langenbruch, M.: Hermite functions and weighted spaces of generalized functions. Manuscripta Math. 119(3), 269–285 (2006)Meise, R., Vogt, D.: Introduction to Functional Analysis. Clarendon Press, Oxford (1997)Petzsche, H.J.: Die nuklearität der ultradistributionsräume und der satz vom kern I. Manuscripta Math. 24, 133–171 (1978)Pietsch, A.: Nuclear Locally Convex Spaces. Springer, Berlin (1972)Pilipović, S., Prangoski, B., Vindas, J.: On quasianalytic classes of Gelfand-Shilov type. Parametrix and convolution. J. Math. Pures Appl. 116, 174–210 (2018)Rodino, L.: Linear Partial Differential Operators in Gevrey Spaces. World Scientific Publishing Co. Inc, River Edge, NJ (1993)Rodino, L., Wahlberg, P.: The Gabor wave front set. Monatsh. Math. 173, 625–655 (2014)Schmets, J., Valdivia, M.: Analytic extension of ultradifferentiable Whitney jets. Collect. Math. 50(1), 73–94 (1999

Collaboration between clinical and academic laboratories for sequencing SARS-CoV-2 genomes

Genomic sequencing of SARS-CoV-2 continues to provide valuable insight into the ever-changing variant makeup of the COVID-19 pandemic. More than three million SARS-COV-2 genomes have been deposited in GISAID, but contributions from the United States, particularly through 2020, lagged behind the global effort. The primary goal of clinical microbiology laboratories is seldom rooted in epidemiologic or public health testing and many labs do not contain in-house sequencing technology. However, we recognized the need for clinical microbiologists to lend expertise, share specimen resources, and partner with academic laboratories and sequencing cores to assist in SARS-COV-2 epidemiologic sequencing efforts. Here we describe two clinical and academic laboratory collaborations for SARS-COV-2 genomic sequencing. We highlight roles of the clinical microbiologists and the academic labs, outline best practices, describe two divergent strategies in accomplishing a similar goal, and discuss the challenges with implementing and maintaining such programs

Homologous Recombination Mediates Functional Recovery of Dysferlin Deficiency following AAV5 Gene Transfer

The dysferlinopathies comprise a group of untreatable muscle disorders including limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment syndrome, and rigid spine syndrome. As with other forms of muscular dystrophy, adeno-associated virus (AAV) gene transfer is a particularly auspicious treatment strategy, however the size of the DYSF cDNA (6.5 kb) negates packaging into traditional AAV serotypes known to express well in muscle (i.e. rAAV1, 2, 6, 8, 9). Potential advantages of a full cDNA versus a mini-gene include: maintaining structural-functional protein domains, evading protein misfolding, and avoiding novel epitopes that could be immunogenic. AAV5 has demonstrated unique plasticity with regards to packaging capacity and recombination of virions containing homologous regions of cDNA inserts has been implicated in the generation of full-length transcripts. Herein we show for the first time in vivo that homologous recombination following AAV5.DYSF gene transfer leads to the production of full length transcript and protein. Moreover, gene transfer of full-length dysferlin protein in dysferlin deficient mice resulted in expression levels sufficient to correct functional deficits in the diaphragm and importantly in skeletal muscle membrane repair. Intravascular regional gene transfer through the femoral artery produced high levels of transduction and enabled targeting of specific muscle groups affected by the dysferlinopathies setting the stage for potential translation to clinical trials. We provide proof of principle that AAV5 mediated delivery of dysferlin is a highly promising strategy for treatment of dysferlinopathies and has far-reaching implications for the therapeutic delivery of other large genes

The Gabor wave front set in spaces of ultradifferentiable functions

[EN] We consider the spaces of ultradifferentiable functions S as introduced by Bjorck (and its dual S) and we use time-frequency analysis to define a suitable wave front set in this setting and obtain several applications: global regularity properties of pseudodifferential operators of infinite order and the micro-pseudolocal behaviour of partial differential operators with polynomial coefficients and of localization operators with symbols of exponential growth. Moreover, we prove that the new wave front set, defined in terms of the Gabor transform, can be described using only Gabor frames. Finally, some examples show the convenience of the use of weight functions to describe more precisely the global regularity of (ultra)distributions.The authors were partially supported by the INdAM-Gnampa Project 2016 "Nuove prospettive nell'analisi microlocale e tempo-frequenza", by FAR2013, FAR2014 (University of Ferrara) and by the project "Ricerca Locale - Analisi di Gabor, operatori pseudodifferenziali ed equazioni differenziali" (University of Torino). The research of the second author was partially supported by the project MTM2016-76647-P.Boiti, C.; Jornet Casanova, D.; Oliaro, A. (2019). The Gabor wave front set in spaces of ultradifferentiable functions. Monatshefte für Mathematik. 188(2):199-246. https://doi.org/10.1007/s00605-018-1242-3S1992461882Albanese, A., Jornet, D., Oliaro, A.: Quasianalytic wave front sets for solutions of linear partial differential operators. Integr. Equ. Oper. Theory 66, 153–181 (2010)Albanese, A., Jornet, D., Oliaro, A.: Wave front sets for ultradistribution solutions of linear partial differential operators with coefficients in non-quasianalytic classes. Math. Nachr. 285(4), 411–425 (2012)Björck, G.: Linear partial differential operators and generalized distributions. Ark. Mat. 6(21), 351–407 (1966)Boiti, C., Gallucci, E.: The overdetermined Cauchy problem for $$\omega$$ ω -ultradifferentiable functions. Manuscripta Math. 155(3-4), 419–448 (2018)Boiti, C., Jornet, D.: A simple proof of Kotake–Narasimhan theorem in some classes of ultradifferentiable functions. J. Pseudo-Differ. Oper. Appl. 8(2), 297–317 (2017)Boiti, C., Jornet, D.: A characterization of the wave front set defined by the iterates of an operator with constant coefficients. Rev. R. Acad. Cienc. Exactas Fs. Nat. Ser. A Math. RACSAM 111(3), 891–919 (2017)Boiti, C., Jornet, D., Juan-Huguet, J.: Wave front sets with respect to the iterates of an operator with constant coefficients. Abstr. Appl. Anal. 2014, 1–17 (2014). https://doi.org/10.1155/2014/438716Boiti, C., Jornet, D., Oliaro, A.: Regularity of partial differential operators in ultradifferentiable spaces and Wigner type transforms. J. Math. Anal. Appl. 446, 920–944 (2017)Bonet, J., Meise, R., Melikhov, S.N.: A comparison of two different ways to define classes of ultradifferentiable functions. Bull. Belg. Math. Soc. Simon Stevin 14(3), 425–444 (2007)Borwein, J.M., Lewis, A.S.: Convex Analysis and Nonlinear Optimization. Theory and Examples, CMS Books in Mathematics/Ouvrages de Mathématiques de la SMC. Springer, New York (2006)Braun, R.W., Meise, R., Taylor, B.A.: Ultradifferentiable functions and Fourier analysis. Result. Math. 17, 206–237 (1990)Cappiello, M., Schulz, R.: Microlocal analysis of quasianalytic Gelfand–Shilov type ultradistributions. Complex Var. Elliptic Equ. 61(4), 538–561 (2016)Carypis, E., Wahlberg, P.: Propagation of exponential phase space singularities for Schrödinger equations with quadratic Hamiltonians. J. Fourier Anal. Appl. 23(3), 530–571 (2017)Christensen, O.: An Introduction to Frames and Riesz Bases. Applied and Numerical Harmonic Analysis. Springer, Berlin (2016)Fernández, C., Galbis, A., Jornet, D.: Pseudodifferential operators on non-quasianalytic classes of Beurling type. Studia Math. 167(2), 99–131 (2005)Fernández, C., Galbis, A., Jornet, D.: Pseudodifferential operators of Beurling type and the wave front set. J. Math. Anal. Appl. 340(2), 1153–1170 (2008)Fieker, C.: $$P$$ P -Konvexität und $$\omega$$ ω -Hypoelliptizität für partielle Differentialoperatoren mit konstanten Koeffizienten. Diplomarbeit, Mathematischen Institut der Heinrich-Heine-Universität Düsseldorf (1993)Gröchenig, K.: Foundations of Time-Frequency Analysis. Birkhäuser, Boston (2001)Gröchenig, K., Zimmermann, G.: Spaces of test functions via the STFT. J. Funct. Spaces Appl. 2(1), 25–53 (2004)Heil, C.: A Basis Theory Primer. Applied and Numerical Harmonic Analysis. Springer, New York (2011)Hörmander, L.: Fourier integral operators. Acta Math. 127(1), 79–183 (1971)Hörmander, L.: Quadratic hyperbolic operators. In: Cattabriga, L., Rodino, L. (eds.) Microlocal Analysis and Applications. Lecture Notes in Mathematics, pp. 118–160. Springer, Berlin (1991)Hörmander, L.: The Analysis of Linear Partial Differential Operators, vol. I. Springer-Verlag, Berlin (1983)Hörmander, L.: The Analysis of Linear Partial Differential Operators, vol. II. Springer-Verlag, Berlin (1983)Hörmander, L.: The Analysis of Linear Partial Differential Operators, vol. III. Springer-Verlag, Berlin (1985)Janssen, A.J.E.M.: Duality and biorthogonality for Weyl–Heisenberg frames. J. Fourier Anal. Appl. 1(4), 403–436 (1995)Langenbruch, M.: Hermite functions and weighted spaces of generalized functions. Manuscripta Math. 119(3), 269–285 (2006)Meise, R., Vogt, D.: Introduction to Functional Analysis. Oxford Science Publications, Clarendon Press, Oxford (1997)Nakamura, S.: Propagation of the homogeneous wave front set for Schrödinger equations. Duke Math. J. 126, 349–367 (2005)Nicola, F., Rodino, L.: Global Pseudo-Differential Calculus on Euclidean Spaces. Springer, Basel (2010)Pilipović, S.: Tempered ultradistributions. Boll. U.M.I. B (7) 2(2), 235-251 (1988)Prangoski, B.: Pseudodifferential operators of infinite order in spaces of tempered ultradistributions. J. Pseudo-Differ. Oper. Appl. 4(4), 495–549 (2013)Pilipović, S., Prangoski, B.: Anti-Wick and Weyl quantization on ultradistribution spaces. J. Math. Pures Appl. 103(2), 472–503 (2015)Rodino, L.: Linear Partial Differential Operators and Gevrey Spaces. World Scientific Publishing Co., Inc., River Edge, NJ (1993)Rodino, L., Wahlberg, P.: The Gabor wave front set. Monatsh. Math. 173, 625–655 (2014)Schulz, R., Wahlberg, P.: Microlocal properties of Shubin pseudodifferential and localization operators. J. Pseudo-Differ. Oper. Appl. 7(1), 91–111 (2016)Schulz, R., Wahlberg, P.: Equality of the homogeneous and the Gabor wave front set. Commun. Partial Differ. Equ. 42(5), 703–730 (2017)Shubin, M.A.: Pseudodifferential Operators and Spectral Theory. Springer-Verlag, Berlin (1987)Sjöstrand, J.: Singularités analytiques microlocales. Astérisque 95, 1–166 (1982)Toft, J.: The Bargmann transform on modulation and Gelfand–Shilov spaces, with applications to Toeplitz and pseudo-differential operators. J. Pseudo-Differ. Oper. Appl. 3(2), 145–227 (2012)Toft, J.: Images of function and distribution spaces under the Bargmann transform. J. Pseudo-Differ. Oper. Appl. 8(1), 83–139 (2017)Treves, F.: Topological vector spaces, distributions and kernels. Academic Press, New York (1967

Safety of AAV Factor IX Peripheral Transvenular Gene Delivery to Muscle in Hemophilia B Dogs

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector–mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4+FoxP3+IL-10+ T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle