Microwire microelectrode arrays (MEAs) have been a popular low-cost tool for chronic electrophysiological recordings and are an inexpensive means to record the electrical dynamics crucial to brain function. However, both the fabrication and implantation procedures for multi-MEAs on a single rodent are time-consuming and the accuracy and quality are highly manual skill-dependent. To address the fabrication and implantation challenges for microwire MEAs, (1) a computer-aided designed and 3D printed skull cap for the pre-determined implantation locations of each MEA and (2) a benchtop fabrication approach for low-cost custom microwire MEAs were developed. A proof-of-concept design of a 32-channel 4-MEA (8-wire each) recording system was prototyped and tested through Sprague Dawley rat recordings. The skull cap design, based on the CT-scan of a single rat conforms well with multiple Sprague Dawley rats of various sizes, ages, and weight with a minimal bregma alignment error . The prototyped 32-channel system was able to record the spiking activities over five months. The developed benchtop fabrication method and the 3D printed skull cap implantation platform would enable neuroscience groups to conduct in-house design, fabrication, and implantation of customizable microwire MEAs at a lower cost than the current commercial options and experience a shorter lead time for the design modifications and iterations. The electrophysiological recording of single neuron activities is critical for understanding the brain functioning mechanisms ,2. To gaSince the brain is composed of multiple interacting regions, multiple MEAs implanted across regions can reveal fast timescale electrical dynamics crucial to brain function. The current approaches for multiple microwire MEA recordings in rodents rely on the repeated single implant craniotomes and other tedious stereotactic-based surgical procedures that are not automated, nor do they take advantage of modern mechanical tools. Specifically, in typical surgeries, the implant locations are individually determined within surgery and a separate craniotomy and durotomy are performed at each location ,21. DuriFabricating MEAs themselves is also a highly manual skill-dependent process. The general fabrication steps involve the electrode alignment which counts on manual work with grids and hands-on small wire soldering ,27,28, aTo enable rodent neuroscience groups to design, fabricate, and implant custom microwire MEAs in house, this study developed (1) a digitally designed and 3D printed custom skull cap with predetermined insertion locations and depths for accurate but simplified multi-region recordings and (2) a benchtop fabrication approach for low-cost, highly customizable, and in-house made microwire-based MEAs using easily accessible materials and tools with minimal training needed. To demonstrate the feasibility and reliability of the developed methodology to provide in-vivo rat brain electrophysiological recordings, a proof-of-concept surgery was planned and conducted to implant four 8-channel planar microelectrode arrays of 50 μm diameter tungsten microwires and 1 mm pitch to record from the cortical areas of a Sprague Dawley rat brain. This 32-channel MEA configuration allows us to span broadly along the anterior-posterior axis of the rat brain and showcase the surgery simplification enabled by the 3D printed custom skull cap.In this section, the design and fabrication approach for our 32-channel 4-MEA recording system is elaborated as a demonstration of our 3D printed skull cap and benchtop microwire MEA fabrication method. The generalization strategy to move beyond this application-specific design is described at the end of each subsection.In this demonstration study, the 32-channel recording headstage  determined the end female connector  that the developed electrical connection system would be connected to. To provide the connections between each of the 32 microwires to one of the pins of the headstage connector, a two-stage PCB system was proposed and developed, as shown in TM connectors shown in The PCB designs and parameters shown in this proof-of-concept trial were tailored, based on the specific recording species and the task in this study. The same concept of a two-stage connection system composed of a planar rigid and flexible PCBs could be implemented with different MEA pitch sizes, various total channel counts and distribution (determining wire number in each MEA), the preferred types of connectors  scan of the rat skull a was firTo evaluate the possibility of using the skull cap design based on a single Sprague-Dawley’s CT scan on different Sprague Dawley rats, a test was conducted aiming to quantify the variation of the bregma location among rats and to create a universal reference point on the headcap design to serve as the bregma reference location. Based on the geometry from the CT scan of a single rat skull, a stereolithography (SLA) 3D printer was used to create an alignment test headcap with six (6) 0.5 mm diameter through-holes separated by 2.2 mm along the anterior/posterior (A/P) direction d. This rFor the proof-of-concept animal surgery in this study, the features to be supported and/or guided by the custom skull cap, included four (4) microwire MEAs and two ground screws. For each MEA, the skull should provide the accurate positions and orientations along the medial/lateral and anterior/posterior directions as well as guidance and stop features along the implantation direction. As shown in As shown in To fabricate the microwire-based MEAs used in this study, 50 µm diameter tungsten microwires with polyimide insulation  were used as the raw material. The wires were hand-cut with fine surgical scissors  into 6 mm long segments and a 2.2 mm long insulation layer was stripped from each segment by a razor blade a. To aliBesides handling and placement, the soldering of the microwires was another labor-intensive and time-consuming step during the MEA fabrication. Worse yet, some microwire materials, such as stainless steel and tungsten, had nonoptimal solderability and would usually require extra steps of surface cleaning, pre-plating, pre-tinning, or specialized soldering ,33,34. TThis modular assembly approach, the 3D printed alignment fixture, and the usage of silver epoxy could significantly lower the time and effort for the MEA fabrication. For an inexperienced operator to build four 8-channel MEAs, it took about 3–4 h, including about 1.5 h of wire trimming, 1.5 h of wire alignment/fixation and connector soldering, and 1 h idle time for the silver epoxy curing. With an experienced operator, the entire process could be completed within 2–2.5 h including the 1 h oven curing time. This stands in significant contrast to the often expensive commercial solutions.To generalize this benchtop MEA assembly approach, depending on the fabrication resources available, the 3D printed alignment fixture with ridges could also be made by precision milling of metal blocks or chemical etching on silicon, to fulfill the different MEA pitch size needs. If the raw microwire material was un-insulated when initially obtained, an extra insulation step  would need to be added at the beginning or after the assembly. If the impedance test yielded a higher value than preferred, an additional conductive coating  could be applied through electroplating . MEAs wiThe MEA assembled, as elaborated in The coordinates of the true bregma positions of six Sprague Dawley rats in the A/P–M/L coordinate system, with the fourth anterior hole in the template cap as origin, are listed in Four MEAs containing eight wires each, were inserted through the headcap, as detailed above. The 32 wires formed a rectangular grid spanning 7 mm along the anterior/posterior axis and 10 mm along the medial/lateral axis to allow for the broad sampling of the cortical activity. All wires were inserted between the coronal and lambdoid skull sutures, to a depth of 1.6 mm. The high-resolution recordings were collected at 20 kHz to obtain the local field potential and single unit signals.The local field potentials (LFP) from 32 channels were recorded over many weeks, during the home-cage sessions, each lasting 2–6 h a. The prAs seen in the results section, the top portions of the skull can be replaced by printed parts without compromising animal health, behavior, or recording quality. We’ve also shown that our cap can be used to insert many wires that yield stable and high-resolution recordings over multiple months. Our CT-aided design conforms well to the skulls of Sprague Dawley rats of varying size, age, and weight, and creates a minimal bregma alignment error. For higher resolution alignment needs, CT head scans (bone) and T1 magnetic resonance imaging (MRI) scan (brain structure) could be co-registered to directly correlate the custom skull cap design with targeted brain structures .In-vivo rat brain recording results from the proof-of-concept study showed the feasibility of the developed design, fabrication, and implantation methodologies to deliver high-quality chronic neural recordings. Most of the methodologies showcased in this proof-of-concept study could be generalized to the electrophysiological recordings and stimulations on the different species, with various devices and configurations, thanks to the capability provided by the benchtop custom MEA fabrication process, CAD, and 3D printing. Because our caps are 3D-printable, they are affordable, widely accessible, and can also be tailored for custom recording locations for specific regions or more broadly for large-scale cortical recordings. This new implantation method could also accommodate other recording designs, including micro-drive manipulators, tetrodes, silicon probes, optogenetic fibers, and imaging windows. Each of these recording options can be planned prior to surgery to reduce the manual manipulation and implantation difficulty, resulting in surgeries that are more efficient and repeatable, and offering high-resolution and high-density and/or broad-scale sampling, and providing stable chronic recordings.Given that brain dynamics involve multi-region coordinations, the ability to better study those dynamics will depend on the development of systems to implant complex arrays of probes that are not practically feasible with manual methods. Computer-based designs and the printing of surgery-enabling platforms is very likely to become increasingly commonplace as rodent brain surgeries become more complex. The developed system represents an important step towards the computer-aided rodent brain implantation to better study the brain and the disease models, from the perspective of broad-scale electrophysiologic dynamics.This study has shown that the development of a new methodology, based on a computer-aided designed and 3D printed skull cap system for both MEA fabrication and rat brain surgery. This work reduces the labor and time for the creation and the surgical implantation of MEAs and allows laboratories to undertake the fabrication on their own, at low cost. The printed skull replacement with the ability to pre-shape and pre-form craniotomies and mounts for surgeries, provides a system that is convenient and effective now, but also is an important first step for future developments.In the developed benchtop fabrication process, while largely improved, the microwire placement and alignment step still requires an extensive labor effort. Furthermore, the impedance result of the assembled MEAs depended upon the wire stripping/cut off and silver epoxy application quality. Future work to further improve the fabrication process will be focused on the machine-based assembly of the microwire MEAs, including the automatic wire feeding and the alignment and machine-controlled epoxy dispensing. For the 3D printed skull cap, future studies will focus on the design considerations to further minimize the bregma location error, along the A/P direction and expansion of the skull cap concept into other rodent species and even primates.A new methodology for laboratories to fabricate a low-cost custom microwire-based MEAs for the multiple brain regions’ implantation, based on a computer-aided and designed 3D printed skull cap platform, has been developed.The skull cap design, based on the CT-scan of a single Sprague Dawley rat, fits the skull of varying sizes, ages, and weight Sprague Dawley rats (n = 6), with standard error of 0.25 mm and 0.07 mm along the A/P and M/L directions, respectively, indicating the feasibility to identify the implantation locations on a general skull cap design, without an animal-specific CT scan and be able to reliably implant the arrays without stereotactic guidance.Based on the proof-of-concept design, the prototype, and the animal recording study, the benchtop fabricated MEAs implanted through the 3D printed skull cap, were shown to be capable of recording spiking activities over multiple months.The design, fabrication, and implantation methodologies elaborated in this study could be duplicated, reconfigured, and/or generalized to various devices and configurations on different species, to enable low-cost highly custom multi-region recording/stimulation studies.


We have come a long way since the start of the COVID-19 pandemic—from hoarding toilet paper and wiping down groceries to sending our children back to school and vaccinating billions. Over this period, the global community of epidemiologists and evolutionary biologists has also come a long way in understanding the complex and changing dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. In this Review, we retrace our steps through the questions that this community faced as the pandemic unfolded. We focus on the key roles that mathematical modeling and quantitative analyses of empirical data have played in allowing us to address these questions and ultimately to better understand and control the pandemic. By February 2020, it was clear that a global coronavirus pandemic had begun to unfold. The rapid rise in COVID-19 cases before lockdown measures went into effect in Wuhan, China, on 23 January 2020 showed how efficient human-to-human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was. By January 2020, the increasing number of documented cases of SARS-CoV-2 outside of China further indicated that regional containment of this virus was going to be extremely unlikely. At this time, epidemiologists and infectious disease modelers began to characterize the key features of the 2019 novel coronavirus (2019-nCoV)—subsequently renamed SARS-CoV-2 in February 2020—that were critical for assessing its containment potential and for projecting its spread at various geographic scales .R0 . These projections relied on statistical modeling approaches, which can provide accurate short-term projections but are likely to fail for longer-term predictions because they do not incorporate the process of transmission . Indeed,As the first wave provided epidemiologists with longitudinal SARS-CoV-2 case data, COVID-19 death data, and virus sequence data, the effect that NPIs had on reducing viral spread started to be quantitatively assessed. These analyses found that NPIs were effective at flattening the curve and even at temporarily reducing reproduction numbers to the extent that circulation levels declined , 19. TheAlthough NPIs were effective at reducing viral spread at the population level, infection risk was not reduced equally across populations. Health care workers and frontline workers  were at particularly high infection risk in April and May of 2020 owing to their close contact with SARS-CoV-2–positive individuals. Many other essential occupations were also found to carry increased risk, including public-facing jobs, such as taxi and bus drivers, and jobs in poorly ventilated and crowded settings, such as factories and meatpacking plants. Crowded living settings, where social distancing was not possible, also carried increased risks for infection . Even wiAs anticipated, the widespread use of NPIs, where implemented, exacted considerable social and economic costs. To facilitate the reopening of the economy, researchers sought to distinguish highly effective NPIs from those that could potentially be discarded. Key to assessing the effectiveness of different NPIs was understanding the routes of viral transmission. How easily did the virus spread between individuals through contaminated surfaces (fomites) compared with direct transmission by aerosols or droplets? Although an early study from April 2020 found that SARS-CoV-2 could remain viable on surfaces, such as plastic, for days , subsequLarge outbreaks detected by contact tracing, coupled with emerging virus sequence data, quickly led to the recognition of substantial transmission heterogeneity between individuals. Studies using data from the first wave found that ~10% of cases were responsible for 80% of secondary infections , 30, a rEpidemiological modeling also pointed toward ways in which surveillance and control efforts could be improved upon to allow for relaxation of broadly applied NPIs. For example, modeling studies showed that traditional contact tracing and digital contact tracing had the potential to slow viral spread in the presence of relaxed lockdown measures , 37. AnoThe initial wave of infections was followed by subsequent waves, reminiscent of those seen in the years after the 1918 influenza pandemic . FactorsTheory would predict that soon after the introduction of a zoonotic pathogen, virus adaptation would result in increased transmission between humans. The rapid spread of the D614G variant [with a glycine residue (G) at position 614 in the spike protein replacing an aspartic acid residue (D)] between March and May of 2020 pointed toward the possibility of virus adaptation occurring  Fig. 3AFig. 3A. R0 . St. StR0 , and the development of transmission-reducing immunity through natural infection followed by its waning. Quantifying the interactions between these factors and their relative roles remains an important area for study, critical for projecting the longer-term dynamics of SARS-CoV-2 , 53.By December 2020, vaccines against SARS-CoV-2 were developed, trialed, and approved for emergency use—a timeline unparalleled in the history of vaccinology. Epidemiological factors facilitated the accelerated timeline of these pivotal trials. With little acquired immunity to SARS-CoV-2 and a high incidence of COVID-19 in mid-2020 when trials were underway, the studies reached their prespecified end points rapidly and ahead of anticipated schedule. As is widely appreciated now, the trials demonstrated excellent protection against COVID-19 and severe outcomes, like hospitalization and death. But questions remained even after the vaccines were authorized for use : How lonMany decisions about the vaccine rollout, including policy-relevant questions such as which groups to target initially, had to be made before such knowledge was available, so epidemiological modeling served a key role in exploring the potential impact of decisions under different assumptions. One initial question was whether to maximize coverage by offering a single dose of a two-dose series to as many people as possible or to maintain fidelity to the trial protocols of two doses (spaced 3 to 4 weeks apart for the mRNA vaccines). Modeling studies, parameterized with trial data that showed apparent protection starting 10 to 14 days after receipt of a first dose, indicated that in the short term, a one-dose strategy is likely preferable for limiting disease at the level of the population . HoweverVaccine-related questions broadened to ones regarding the extent to which vaccination could reduce transmission . VaccineComplicating the assessment of SARS-CoV-2 vaccine efficacy was the evolution of new variants of concern around the time that vaccines were rolled out. In late 2020, the B.1.1.7 lineage [now known as Alpha in the World Health Organization’s (WHO’s) variant naming scheme] was detected and rapidly spread in the UK. Several of the mutations harbored by this lineage significantly altered the viral phenotype, including enhanced binding to angiotensin-converting enzyme 2 (ACE2) , with coSoon after the emergence of the Alpha lineage, a different viral lineage, B.1.617.2 , was fueling a large wave of SARS-CoV-2 infections in India . This liIn November 2021, researchers in South Africa noticed a rising number of COVID-19 cases with S gene target failure (SGTF), indicative of an expanding viral subpopulation harboring the Δ69–70 spike gene deletion. Shortly thereafter, circulation of a new variant was confirmed through whole-genome sequencing. Based on its mutational profile and its extremely rapid spread in South Africa, the WHO designated this variant as one of concern, naming it Omicron (B.1.1.529). In December 2021, this variant spread rapidly throughout the world, resulting in more confirmed infections than ever before  and rapiThe emergence of Omicron, and Alpha before it, raises important evolutionary questions regarding the sources of new, genetically divergent variants. At their time of emergence, both variants harbored more mutations than would have been expected given the rate at which SARS-CoV-2 evolves in the human population. Omicron’s distant evolutionary relationship to other SARS-CoV-2 variants is notable . One posSARS-CoV-2 has been difficult to control since the start of the pandemic, and the recent spread of Omicron has only amplified this challenge. It is inevitable that SARS-CoV-2 will become an endemic pathogen in the human population. However, its impact on our health and daily lives will change considerably over time . GovernmThe most evolutionarily successful variants of SARS-CoV-2 so far have evolved greater transmissibility relative to more ancestral variants. Although higher transmissibility will result in more infections, increasing population-level immunity from infections and vaccinations might make these infections less severe in terms of health impact. However, the evolutionary pressures facing the virus, and thus its patterns of adaptive evolution, will change. Variants, such as Omicron, that gain much of their transmission advantage from evading immunity may become the norm, as is the case with seasonal influenza.The evolution of SARS-CoV-2 virulence in terms of how harmful or deadly it is has been a topic of debate since the beginning of the pandemic . EvolutiThe most pressing scientific questions have changed over the past 2 years, reflecting the complexity of this pandemic . Early q


Translocation of chloroplast-located genes to mitochondria or nucleus is considered to be a safety strategy that impedes mutation of photosynthetic genes and maintains their household function during evolution. The organelle translocation strategy is also developed in photosynthetic NDH-1 (pNDH-1) genes but its understanding is still far from complete. Here, we found that the mutation rate of the conserved pNDH-1 genes was gradually reduced but their selection pressure was maintained at a high level during evolution from cyanobacteria to angiosperm. By contrast, oxygenic photosynthesis-specific (OPS) pNDH-1 genes had an opposite trend, explaining the reason why they were transferred from the reactive oxygen species (ROS)-enriched chloroplast to the ROS-barren nucleus. Further, genome-wide sequence analysis supported the possibility that all conserved pNDH-1 genes lost in chloroplast genomes of Chlorophyceae and Pinaceae were transferred to the ROS-less mitochondrial genome as deduced from their truncated pNDH-1 gene fragments. Collectively, we propose that the organelle translocation strategy of pNDH-1 genes during evolution is necessary to maintain the function of the pNDH-1 complex as an important antioxidant mechanism for efficient photosynthesis. All com1) genes , which i1) genes . They co1) genes . It is f1) genes  but its 1) genes . However1) genes . HoweverHere, we calculated and analyzed the mutation frequency and selection pressure, explaining the reason why these OPS pNDH-1 genes were transferred from chloroplast to the nucleus. Further, we found the presence of conserved pNDH-1 gene fragments in mitochondrial genomes of Chlorophyceae and Pinaceae, implying that these conserved pNDH-1 genes lost in chloroplast genomes of Chlorophyceae and Pinaceae were transferred to their mitochondrial genomes. Collectively, our data provide new insights into the organelle translocation of pNDH-1 genes during evolution from cyanobacteria to angiosperm.rbcL gene of Methanogen, Cyanobacteria, Chlorophyceae, Bryophyta, Pinaceae, Monocots, and Dicots. The gene sequences of rbcL from different species were obtained from the National Center for Biotechnology Information (NCBI1). The names of the selected species and their GenBank accession numbers are listed in 2) was used for the unrooted phylogenetic tree visualization.Phylogenetic tree was constructed based on dS) and non-synonymous substitution rate (dN) were calculated using DNAsp6 software  was carried out with the aid of a pipeline SAMEM v.0.83.3 The SAMEM package . Eleven 4 and all GenBank accession numbers are listed in The data underlying this article are available within the NCBI GenBank database,Genome-wide sequence analysis suggests that pNDH-1 originates from a group 4 membrane-bound [NiFe] hydrogenase  and evoldS  genes was gradually decreased but their selection pressure was maintained at a relatively high level during evolution from methanogen to dicots pink in . HoweverDuring the evolution from prokaryotic cyanobacteria to eukaryotic angiosperm, organelle translocation is considered to be an important safety strategy that impedes mutation of photosynthetic genes and maintain their household function . Such orvia decreasing their mutation rate and increasing their selection pressure, while OPS pNDH-1 genes develop another safety strategy via transferring them from the chloroplast genome to a relatively safe nucleus genome (The results of this study found that two distinctly different strategies have been developed by conserved and OPS pNDH-1 genes to impede their mutations and maintain their functions . Conservs genome . It is ws genome , gene reConsistent with the previously reported Orchidaceae , 2017, t2. Consistent with the hypothesis, Pinaceae underwent an O2-rise phase (2-enriched chloroplast to the O2-less mitochondria as oxygen-consuming organelle.Compared with the land plants, Bryophyta and Pteridophyte, the trees in Pinaceae grow quite high , implyinse phase  and may se phase . As a coBased on the aforementioned analysis, we propose that translocation of pNDH-1 genes from chloroplast genome to the nucleus genome or mitochondrial genome is important to maintain the architecture and household function of pNDH-1 during evolution. As a consequence, the function of pNDH-1 as an important antioxidant mechanism can reduce ROS production necessary for the survival of eukaryotic photosynthetic organisms in aerobic environment.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.WM conceived and designed the project. JY and JZ performed the bioinformatics analysis. JY, ZR, LW, and WM interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hierarchical guidance is developed for three-dimensional (3D) nanoscale X-ray imaging, enabling identification, refinement, and tracking of regions of interest (ROIs) within specimens considerably exceeding the field of view. This opens up new possibilities for in situ investigations. Experimentally, the approach takes advantage of rapid multiscale measurements based on magnified projection microscopy featuring continuous zoom capabilities. Immediate and continuous feedback on the subsequent experimental progress is enabled by suitable on-the-fly data processing. For this, by theoretical justification and experimental validation, so-called quasi-particle phase-retrieval is generalised to conical-beam conditions, being key for sufficiently fast computation without significant loss of imaging quality and resolution compared to common approaches for holographic microscopy. Exploiting 3D laminography, particularly suited for imaging of ROIs in laterally extended plate-like samples, the potential of hierarchical guidance is demonstrated by the in situ investigation of damage nucleation inside alloy sheets under engineering-relevant boundary conditions, providing novel insight into the nanoscale morphological development of void and particle clusters under mechanical load. Combined with digital volume correlation, we study deformation kinematics with unprecedented spatial resolution. Correlation of mesoscale (i.e. strain fields) and nanoscale (i.e. particle cracking) evolution opens new routes for the understanding of damage nucleation within sheet materials with application-relevant dimensions. But often the possibilities for sample miniaturisation are limited, e.g. by the need for preserved boundary conditions or the danger of local disintegration by sample extraction. They also cannot keep up with the correspondingly reducing field of view (FOV) related to the increasing spatial resolution enabled by the progress of X-ray microscopy. For this reason, local 3D imaging approaches like local computed tomography (CT)1 or computed laminography (CL)3 play an increasingly important role for many applications related. For these local techniques, however, the identification and selection of possible regions of interest (ROIs) within the much larger samples is often hindered or completely precluded by the excessive superposition of features in the detected two-dimensional (2D) projection images. This is further complicated for in situ studies, where samples may undergo additional morphological changes or displacements. In consequence, new measurement strategies and techniques are required for the identification and continuous readjustment of the imaged sample sub-volume containing the features of interest. In this context, a serious complication arises from the formation of Fresnel diffraction patterns in the recorded images, caused by the propagation of the X-ray wave front from the sample to the detection plane. This in particular applies to magnified projection microscopy where the inherent conical beam effectively enhances the impact of Fresnel diffraction on the detected image. Consequently, in most cases a direct interpretation of the measured raw data as well as of 3D reconstructions based directly on raw data is not possible without prior image processing by means of suitable phase-retrieval algorithms.Modern X-ray microscopy allows the non-destructive three-dimensional (3D) investigation of samples at nanoscale resolutionWe here introduce so-called hierarchical guidance for 3D X-ray microscopy, overcoming the above limitations of 3D nano-imaging by enabling the identification and refinement of the inherently small ROIs inside considerably larger samples. Particularly for in situ imaging experiments aiming on such high spatial resolution, accurate ROI tracking is considered a prerequisite. In short, as illustrated in Fig. 4, which allows for a flexible adjustment of the magnification and related FOV by translating the sample between focal spot and detection plane5, see Fig. 7. For the realisation of the crucial on-the-fly (i.e. sufficiently fast) phase-retrieval suited for high-fidelity and high-resolution imaging , we extend the so-called quasi-particle (QP) approach9 to a conical beam. For this, we carefully justify this extension theoretically, followed by a validation by simulations as well as by application to experimental data. In particular, we show that the obtained phase reconstruction of the magnified holographic data  does not suffer significantly regarding image quality compared to established multi-distance5 and/or iterative10 phase-retrieval approaches, all of which are considerably more time-consuming regarding measurement and/or phase-retrieval computation, respectively, and therefore not well applicable here. A similar on-the-fly capability like the extended non-iterative single-distance QP phase-retrieval is currently only achieved for data acquired in the edge-enhancement regime13, but which does not provide sufficient spatial resolution for our purposes. After phase-retrieval, the obtained phase images are directly streamed to a state-of-the-art GPU-based 3D tomographic or laminographic reconstruction pipeline12, resulting in a total data processing time sufficiently short for true on-the-fly execution. This means that directly at the end of the experimental data acquisition, or only with negligible delay, a picture of the internal sample morphology is accessible as overview or for browsing in detail, enabling immediate feedback for decision-making regarding subsequent experimental steps.The required zoom capability down to the nanoscale is realised by means of projection microscopyIn order to demonstrate the potential of the proposed hierarchically guided X-ray nanolaminography, we apply this approach to enable in situ 3D imaging with deep sub-micrometre spatial resolution for studying damage nucleation during ductile fracture inside an aluminium alloy sheet (Al-Cu-Mg AA2139T3)) under plastic deformation. Here only sub-micrometre spatial resolution allows observing the relevant void formation and secondary phase particle cracking mechanisms, while in the same time a macroscopic sample size is indispensable for the realisation of the strain state required, see Fig. 39T3) und14. Here new experimental insight and corresponding models are needed to be able to predict the effect of particle content on mechanical properties. Particle nature, size and distribution are also known to play an important role in this process15. For the development of such new models, the knowledge of the in situ microstructural development (including voids and secondary phase particles) as well as its correlation to the strain magnitude and conditions within the material is crucial. Previous studies on damage nucleation and growth have been performed in situ at the microscale17, but only ex situ at the nanoscale20. Here the study presented opens new possibilities for obtaining a comprehensive picture of damage processes in materials via the combination of 3D nanoscale resolution and in situ measurement. Thereby, hierarchical guidance is key for identifying, zooming-in-on and tracking damage-relevant clusters of voids and secondary phase particles in situ, with high resolution, and still within sufficiently large samples maintaining the application-relevant boundary conditions.With increasing environmental and economical demand, recycling and circular industries become of interest for alloy manufacturers, in particular for aluminium alloys. However, recycling of alloys of different composition may result in an increase of impurity and associated intermetallic particle content that in turn may affect important mechanical properties like the formability21 plays a crucial role for technological applications of the investigated material and is typically split into three characteristic steps: void nucleation, void growth and void coalescence. Continuum damage models are known to describe and predict well the void growth and coalescence phase23, while usually only phenomenological models are available for the void nucleation phase. The latter depict the void nucleation rate as a Gaussian function having a maximum rate at a critical strain24. However, the contributions of stress and strain to the nucleation process are currently not sufficiently well known. Moreover, all these models cannot account for morphology, size, and nature of particles. We here show the feasibility of analysing nanoscale morphology and of measuring displacements via digital volume correlation (DVC) based on the obtained multiscale data, in this way enabling the correlation of mesoscale strained bands and nanoscale damage nucleation processes.Moreover, ductile fractureIn the following, first the concept and principles of hierarchical guidance are introduced. For its implementation, the adaptation of QP phase-retrieval to the conical beam case plays a central role and is thus described in more detail. In particular a theoretical and experimental justification of its validity under the changed scaling conditions is given and the image quality is compared to established and commonly used phase-retrieval approaches for X-ray microscopy. In the second part, we demonstrate the application of the approach for the observation of an AA2139T3 aluminium alloy sheet during in situ mechanical testing. Here the main aspects of the experimental setup for the in situ loading test are described, followed by the nanoscale results obtained in combination with a DVC strain analysis.The experimental workflow of hierarchically guided X-ray microscopy is illustrated in Fig. 1, its large energy bandwidth enables sub-second exposure times , which is a direct consequence of the employed single step phase-retrieval algorithm11. This algorithm is not applicable to holographic data obtained by X-ray projection microscopy. On the other hand, all published phase-retrieval approaches suited for projection microscopy require time-consuming multi-distance acquisitions25 or iterative procedures26, which allow compensating for non-measured frequencies and overcoming certain limitations of the underlying physical models. The multi-distance approaches25 require time-consuming measurements, the computation of several Fourier transforms and prior image registration. Depending on the number of iterations necessary27, the iterative approaches require the computation of ten to several thousands Fourier transforms, even with state-of-the art hardware resulting in typical reconstruction times of not less than 10.On the one hand, until now all published pipelines capable of on-the-fly phase-retrieval are limited to data recorded in the edge-enhancement regime9 to a diverging cone-beam geometry. The resulting phase-retrieval technique requires the calculation of only a single frequency filter in Fourier space and is applicable to a broad range of samples, since the restrictions of so-called contrast-transfer-function based approaches5 concerning the samples optical properties are considerably relaxed. So far, however, the QP approach has been applied only to parallel beam conditions and thus the resulting imaging resolutions have been limited to about 28). By extending its application to cone-beam geometries the limitation in resolution is reduced to focal spot sizes of state-of-the-art focusing devices29. In the following, the cone-beam adapted version of the QP approach is built on the Fresnel scaling theorem30, but due to the strongly reduced Fresnel numbers the Fourier space coverage for single-distance acquisition changes drastically for a cone-beam compared to parallel beam conditions. Thus, we finally justify the application of the QP approach by a careful comparison to phase-retrieval approaches established for projection microscopy.We here overcome these current speed limitations for high-quality phase-retrieval in the holographic regime by adapting the non-iterative, single-distance QP approach8 is closely related to contrast-transfer-function (CTF) approaches31, which are based on certain assumptions on the optical properties of the sample under investigation (i.e. homogeneous or pure phase object). As a consequence, the QP approach involves a term which regularises frequencies that are corrupted by the violation of these assumptions8. The cone-beam adapted version of the QP approach results in the following relationship between the phase shift A is given byA allows suppressing frequencies where second order phase and attenuation contributions start to be dominant and thus corrupt the inversion of Fresnel propagation (i.e. influences of large phase shifts and absorption). As a result, this approach allows for the imaging of samples with optical properties not suitable for conventional CTF phase-retrieval due to considerably large phase variations, e.g. the here investigated 33, i.e.1M. Thus, in contrast to parallel beam propagation, already for relatively short propagation distances The QP approachentclass1pt{minimaentclass1pt{minima26, see Fig. 11) as input for an iterative optimisation procedure between forward calculated, reconstructed phase maps and measured Fresnel diffraction patterns for several propagation distances (using 10 iterations). (ii) conventional multidistance CTF approach under the assumption of a homogeneous  multidistance acquisition taking into account all non-linearities (strong phase objects and strong absorption) of Fresnel propagationOur comparison of the non-iterative CTF-based multidistance phase-retrieval (ii) in Fig. 2, providing both high resolution and on-the-fly capability as required for the hierarchically guiding approach introduced above.To summarise, the presented comparison of available phase-retrieval approaches shows that the cone-beam version of the QP approach is superior to the alternative single distance approach (iii) regarding the achievable resolution and image quality. The two alternative methods (i) and (ii), which are competitive in terms image quality are iterative and/or multidistance techniques, which are considerably more time consuming during measurement and/or reconstruction. The latter are thus not suited for fast or even on-the-fly computations. In contrast, the cone-beam version of QP turns out to be applicable for the projection data acquired by magnified projection microscopyIn the following, the introduced hierarchical guidance plays a key role in the feasibility study of nanoscale in situ investigations of damage nucleation and growth in an AA2139 T3 aluminium alloy sheet during ductile fracture. The knowledge of these processes are highly relevant for understanding of the dependence of ductile fracture on the material nanostructure and strain conditions. This type of studies have so far been prevented by the lack of nanoscale in situ morphological data of such materials under mechanical loading.34 was used, shown in Fig. The nanolaminography measurements have been acquired with the setup shown in Fig. mentclass2pt{minimvia digital image correlation (DIC) . Thus, the effective pixel size of the detector was equal to For the acquisition of scans with variable spatial resolution the slightly diverging cone-beam provided by the KB-mirror system of ID16b at the ESRF was employed (Fig. 3. However, due to the different acquisition geometry CL achieves superior image quality7. The specific Fourier space sampling results in a high in-plane resolution and characteristic artefacts along the rotation axis38. Laminography has been broadly applied with micrometer spatial resolution e.g.39. Few CL experiments at the nanoscale have been performed, all of them at energies less than 7. Recently, scanning nanolaminography41 has been introduced at very high spatial resolution and outstanding hierarchical capabilities, but at the expense of long scan times, low energies and reduced sample thicknesses.CL is a generalisation of CT, which allows for 3D investigation of flat and laterally extended objects. The rotation axis is inclined by the laminography angle mentclass2pt{minimvia DIC from a speckle pattern painted on sample surface35. The optical microscopy images have a pixel size of 42 is applied to the X-ray CL data sets. The method has been broadly applied to such materials at the micron scale and enabled internal 3D strain fields to be measured16. To obtain results with the sparse feature density of high-resolution scans mechanical regularisation is used43. Using an increased regularisation length (2D strain fields at the surface are assessed


Mycobacterium tuberculosis (Mtb) infection or the progression of the disease. The development of a new vaccine against tuberculosis arises a nontrivial scientific challenge caused by several specific features of the intracellular lifestyle of Mtb and the ability of the pathogen to manipulate host immunity. The purpose of this review is to discuss promising strategies and the possibilities of creating a new vaccine that could replace BCG and provide greater protection. The considered approaches include supplementing mycobacterial strains with immunodominant antigens and genetic engineering aimed at altering the interaction between the bacterium and the host cell, such as the exit from the phagosome. Improved new vaccine strains based on BCG and Mtb undergoing clinical evaluation are also overviewed. Tuberculosis is one of the most threatening infectious diseases and represents an important and significant reason for mortality in high-burden regions. The only licensed vaccine, BCG, is hardly capable of establishing long-term tuberculosis protection and is highly variable in its effectiveness. Even after 100 years of BCG use and research, we still cannot unequivocally answer the question of which immune correlates of protection are crucial to prevent  Mycobacterium tuberculosis (Mtb), is one of the major reasons of death around the world. A total of 1.6 million people died from TB in 2021 (WHO). This disease is characterized by slow progression; after pathogen penetration and primary infection, latent TB usually develops. In this case, the bacteria are confined in granulomas under immune control to restrict bacterial spreading [Tuberculosis disease (TB), which is caused by BCG , the only TB vaccine available today, provides a certain level of defense, especially in children, but it has some drawbacks as well. Advanced variants of the vaccines offering increased immunogenicity and alternative vaccines based on other species of mycobacteria have long been under development all over the world.Whole-cell vaccines, especially live ones, appear to be preferred to subunit vaccines . Their aDue to the emergence of new methods of genetic engineering, the improvement of BCG has received a second wind. Numerous variants of new recombinant vaccine strains and subunit vaccines are being actively developed. This review first covers some aspects of the interaction of mycobacteria with the host cells. A discussion of some popular strategies for modifying BCG to enhance its vaccine properties follows, and attention is given to the most successful vaccine candidates that are currently in clinical trials.–host cell interaction greatly affects both the disease progression and immune response. These points are important for understanding the action of vaccines, especially live vaccines as BCG. Immune response to Mtb comprises different mechanisms of the innate and adaptive immune response. A characteristic feature of Mtb is its ability to live inside professional phagocytes, which normally eliminates the captured bacteria and activates cells of adaptive immunity. In this connection, the MtbMycobacteria are intracellular pathogens; they invade alveolar epithelial cells, macrophages, and other phagocytes. When phagocytes engulf Mtb, the mechanisms aimed at destroying the pathogen are triggered, but Mtb may interfere with them and use the engulfing cell as a habitat. Possible options for the fate of the engulfed mycobacterium are shown in Inhibiting phagosome maturation in macrophages ensures the efficient intracellular multiplication of mycobacteria, especially at the beginning of infection. Mtb products involved in phagosome maturation arrest include cell wall components such as lipoarabinomannan (LAM) , glycoliAnother pathway for Mtb survival in the phagocyte cell is the destruction of the phagosome membrane and its exit into the cytoplasm . The maiSince Mtb is able to avoid death in the phagolysosome, autophagy plays an important role in bacterial elimination. Autophagosomes are two-membrane structures surrounding cell components or intracellular pathogens . The fusAn important consequence of mycobacteria entering the cytosol of the host cell is their interaction with cytosolic innate immunity receptors, leading to the activation of the inflammasome . InflammCytokines of the IL-1 family play a critical role in the protection against TB. IL-1R knockout in mice led to early lethality after aerosol low-dose infection with Mtb . At the The mechanisms of cell death following Mtb infection have different consequences for the host. In classical necrosis, the affected cell swells and bursts, causing acute inflammation. The leakage of cell content during necrosis promotes the spread of bacteria . ApoptosAdaptive immunity is important for the containment of the infection. It is mainly mediated by T-cells. The activation of naive T-lymphocytes occurs in lung-draining lymph nodes where dendritic cells and, to a lesser extent, macrophages locate and present antigens as processed epitopes in complex with MHC I or MHC II . The maiSpecific CD4 T-cells can form different populations. Th1 lymphocytes are critical as major producers of IFN-γ, TNF-α, and IL-2. The genetic dysfunction of IFN-γ leads to the high sensitivity of mycobacteria . The cytThere is no unequivocal opinion on the role of humoral immunity in the protection against the development of TB. It is known that patients with a defective antibody production mechanism and/or patients deficient in B-lymphocytes are not particularly at risk of TB infection, and passive immunization provides no protection. However, several groups have found that monoclonal antibodies against certain cell wall components can modify various aspects of mycobacterial infections . Thus, tMycobacterium bovis in poor media, leading to the loss of several gene loci. The latter are known as regions of difference (RD) and include some sequences that are important for virulence (primarily RD1). A number of other gene rearrangements occurred during subsequent cultivation, resulting in the appearance of different sub-strains, which have some differences in the level of protectiveness and residual virulence [The only vaccine currently used in clinical practice is BCG. It was obtained through the long-term cultivation of pathogenic irulence .The BCG vaccination is administered in infancy in high-burden countries. BCG protection from disseminated TB and tuberculous meningitis, the two most severe forms of TB in children, is reliably proven . In addiAn analysis of current epidemiologic data on COVID-19 infection revealed a correlation between BCG vaccination and decreased morbidity and mortality from COVID-19 worldwide . It was The intradermal BCG vaccination of infants has been found to produce functions specific to mycobacteria CD4 and, to a lesser extent, CD8 cells . BCG proBCG vaccination can protect against infection with Mtb. The meta-analysis of six studies (n = 1745) showed the effectiveness of BCG-induced protection against infection was 27% compared with 71% against active TB. Among those infected, protection against disease progression was 58%. The effectiveness of the protection against infection among vaccinated children based on 14 studies was 19% compared to unvaccinated children .A key problem with BCG is its varying efficacy. Additionally, BCG has little or no effect on the spread of pulmonary TB in adults. Geographic location, prior exposure to nontuberculous mycobacteria, and the rate of attenuation of BCG are considered factors that influence the effectiveness of BCG . TherebyOne promising strategy for improving BCG appears to be the introduction of TB antigens whose expression will enhance the immune response. To date, quite a few antigens have been described that can be used for this purpose.pe35 (Rv3872), esxB (Rv3874), which encodes CFP-10, and esxA (Rv3875), which encodes ESAT-6. The other significant genes are Rv3868, Rv3869, Rv3870, Rv3871, and Rv3877, which encode proteins critical for CFP-10 and ESAT-6 secretion, including chaperone-like proteins that contain an ATP-binding site and probable membrane proteins. They appear to be involved in the formation of the transmembrane complex that enables ESAT-6 and CFP-10 translocation. Extended RD1 also contains several genes, such as Rv3865 and Rv3866, the inactivation of which has not resulted in the damaged expression or impaired secretion of ESAT-6 and CFP-10. Their exact role remains unknown; perhaps they contribute to Mtb virulence independent of the ESAT-CFP secretion system.The attenuation of BCG is caused by the loss of several regions of difference in its genome. The most important of them is RD1, which contains genes encoding ESX-1 components. The major proteins of this system, ESAT-6, and CFP-10, are important Mtb virulence factors that are secreted into the cytosol ,49. A stThese important antigens can be used to create new recombinant vaccine strains (rBCGs). For example, the immune response to the rBCG strain expressing the ESAT-6 secretory antigen, both humoral and cellular, was higher than that of BCG in a mouse model . Many prESAT-6 is often used in vaccines in combination with other antigens, such as Ag85B. For instance, rBCG-AEI expresses a chimeric Ag85B-ESAT-6-IFN- γ protein. This gave it greater immunogenicity compared to the BCG control strain and even compared to recombinant strains expressing these antigens alone .To enhance the vaccine properties of BCG, ESAT-6 is combined with other components related to its expression and secretion, such as proteins of the PE/PPE family. This group is specific for mycobacteria and has similar conserved Pro-Glu and Pro-Pro-Glu motifs, respectively. It includes a large variety of proteins, many of which act as secreted antigens. Some PE and PPE genes are part of the RD1 region and are associated with the ESX secretion system .fbpA, fbp B, and fbpC2 genes, respectively; the major secreted antigen is Ag85B [One of the major virulence factors of Mtb is the Ag85 complex, which consists of three proteins, Ag85A, Ag85B, and Ag85C, encoded by the is Ag85B . Ag85 plis Ag85B . In addiis Ag85B . Additiois Ag85B . All theis Ag85B .A recombinant BCG strain has been engineered that overexpresses the most important Sec proteins SecD, SecF, and SecG . Thus, tMycobacterium smegmatis serves as a common model for research on mycobacteria, including Mtb, because this organism is nonpathogenic and relatively fast-growing. In addition, attempts are being made on the basis of M. smegmatis to create a TB vaccine that can replace BCG. Thus, a recombinant rMs064 vaccine in which M. smegmatis expresses epitopes of the Ag85B protein has been considered. There have been several studies of this vaccine [The Mtb-related  vaccine  in a mouMycobacterium kansasii microorganism, which has great similarity to Ag85B Mtb. Its use in combination with a booster in the form of a plasmid expressing the Ag85B gene (DNA-Mkan85B) showed an enhanced CD8+ T-cell response to Mtb. This study also identified two novel MHC I (H2-Kd)-restricted epitopes that induce cross-reactivity against mycobacteria, including Mtb.The rBCG-Mkan85B vaccine  was alsoThe alpha-crystallin protein HspX, a member of the heat shock protein family, is associated with the cell wall. Under the conditions of the lack or absence of oxygen, mycobacteria change and thicken the cell wall, which occurs with the direct participation of HspX. This allows the cells to survive under anaerobic or microaerobic conditions, which is important for penetration into macrophages . This prA recombinant BCG strain overexpressing the HspX antigen (rBCG::X) was tested in an in vivo model . It exprIn addition to recombinant live vaccines, HspX is often used successfully in subunit vaccines, either as a single antigen or in combination with others ,69,70,71Another strategy is to modify the interaction between mycobacteria and host cells in order to stimulate the signaling pathways leading to the most effective immune response. Often researchers combine this strategy while expanding the antigenic repertoire of the vaccine to maximize efficacy.Cyclic di-AMP (c-di-AMP) is a widely used secondary messenger in bacteria . It is cThe role of c-di-AMP in Mtb virulence is under discussion, and the data from studies in this area are contradictory. On the one hand, increased c-di-AMP levels have been shown to be associated with the attenuation of Mtb virulence . On the disA c-di-AMP causes a significant c-di-AMP-mediated increase in IL-6, IL-1β, IFN regulatory factor 3, and IFN-β [M. smegmatis model [Recombinant BCG with the overexpression of the endogenous mycobacterial diadenylate cyclase gene nd IFN-β . In thisis model .As discussed above, mycobacteria can actively interfere with the intracellular processes of phagocytes and manipulate the immune response. It has been suggested that the lack of efficacy of BCG may be related to its immunosuppressive properties .secA2 in Mtb resulted in partial growth attenuation in vivo. When used as a vaccine, the strain increased the priming of antigen-specific CD8+ T-cells in vivo and reduced bacterial load in the organs of mice and guinea pigs compared with BCG during the Mtb challenge. The authors suggest that an important contribution to the protective properties is made by the preferentially apoptotic type of the death of mutant-infected macrophages. They found out that this effect was dependent on mycobacterial superoxide dismutase A (SodA). SodA is secreted by Mtb through the SecA2 secretion system. It catalyzes the conversion of superoxide anions into hydrogen peroxide. [secA2 and extracytoplasmic-function sigma factor (SigH) was engineered in order to evaluate the role of mycobacterial antioxidants. SigH controls the expression of several antioxidants, including thioredoxin. The modified vaccine had greater immunogenicity than BCG. It demonstrated a greater number of cytokine-producing CD8+ lymphocytes at the peak of the primary immune response and a greater number of IL-2-producing CD4+ lymphocytes [Deletion of eroxide. . Reactiveroxide. ,84. A BCphocytes .The construction of strains deficient in genes whose products are associated with the disruption of phagosome maturation has been suggested as a direction for obtaining new BCG and Mtb vaccine strains.Zmp1 from Mtb is a virulence factor linked to the stopping of phagosome maturation and preventing inflammasome activation . In addizmp1 deletion mutant was more immunogenic in the mouse model. The zmp1 deletion did not affect the survival of BCG-infected mice with severe combined immunodeficiency (SCID) or the growth and distribution of BCG in immunocompetent mice [zmp1 deletion showed better protection by reducing the bacterial load in the lungs of infected guinea pigs. Additionally, unmarked BCG zmp1 mutant strains showed a better safety profile in the SCID mouse survival model than the original BCG strains, which is important because HIV is common in regions with a high TB burden as well [The deletion of Zmp1 in BCG affected the bacterial ability to stop phagosome maturation and resulted in the increased presentation of MHC-restricted class II antigens by dendritic cells compared with BCG. The ent mice . A prote as well .Secretory acid phosphatase SapM may be involved in disrupting phagosome-lysosome fusion by removing of phosphatidylinositol 3-phosphate (PI3P) from phagosome membranes which is necessary for the process. The deletion of SapM in Mtb resulted in the increased maturation of Mtb-containing phagosomes in THP-1 cells and decreased virulence in a guinea pig model . HoweverMycobacterium marinum (BCG::ESX-1Mmar) was constructed. Vaccination with BCG::ESX-1Mmar activated the cGas/STING/TBK1/IRF-3 pathway for type 1 interferon induction and enhanced AIM2 and NLRP3 inflammatory activity, resulting in a higher proportion of specific CD8+ T cells and polyfunctional CD4+ Th1-cells. The strain provided better protection than parental BCG, although the fact that the strain expressed ESX-1-specific immunodominant antigens probably also contributed [A promising approach to switching the immune response is the obtaining of BCG strains that are capable of penetrating into the cytoplasm of cells. The complementation of BCG with the genes coding ESX-1 that we have already discussed above provides the possibility of the rupture of the phagosome, as well as attaining a response to the immunodominant antigens ESAT-6 and CFP-10. However, an increase in virulence is also possible. To avoid this, a recombinant BCG strain supplemented with ESX-1 of the cold-blooded animal pathogen tributed .The idea of returning BCG to its ability to penetrate cytoplasm appears to be quite successful, as evidenced by the fact that vaccines in which this has been implemented have already participated in clinical trials, and one of them is already in phase III (details below).We will introduce the most promising recombinant live vaccines under clinical evaluation .Clostridium perfringens. AERAS 422 also contains plasmid encoding immunodominant antigens expressed by Mtb both during active infection  and during the reactivation of latent infection (Rv3407). This vaccine has shown improved protectivity and comparable safety compared to BCG in preclinical studies [AERAS 422 is a live attenuated BCG-derived vaccine. AERAS 422 cells possess the ability to perforate endosomal membranes due to the expression of PFOG137Q, allowing antigens to penetrate into the cytosol and, thus, stimulating an MHC class I-restricted immune response. PFOG137Q is a gene encoding a mutant perfringolysin (PFOG137Q) from  studies . However studies .Listeria monocytogenes [Another TB vaccine currently in clinical trials is rBCG ΔureC::hly, or VPM1002 . It was ytogenes . This stytogenes .L. monocytogenes is one of the few microorganisms that leave the phagosome and enter the cytoplasm, which promotes the presentation of MHC-I-restricted epitopes [epitopes . An impoureC urease gene from the BCG genome. This way, the pH was lowered to an optimal value of 5.5 for Hly, while macrophage apoptosis was also promoted [This strain was further improved by deleting the promoted . The resrBCG ΔureC::hly is currently in clinical trials. According to the results of phase I clinical trials conducted in Germany and South Africa, VPM1002 was found to be safe and immunogenic. A randomized Phase IIa clinical trial in healthy infants in South Africa showed that the strain exhibited the necessary immunogenicity in the subjects and was well tolerated. Phase IIb was conducted in healthy and HIV-negative infants . A phaseIn addition, a phase III clinical trial involving older adults evaluated the efficacy of this candidate vaccine for the prevention of severe respiratory disease, including COVID-19 . As in pM. bovis. The M. bovis strain is not normally transmitted between humans, but an IS6110 insertion found in the epidemic strain increased the virulence of M. bovis. This mutation resulted in increased transcription of the phoP gene, which is part of the PhoP/PhoR two-component system [phoP mutants can produce but are unable to export ESAT-6 [Another example of the vaccine strains in phase III clinical trials is the MTB-derived strain MTBVAC with two inactivated virulence genes. Researchers chose to base the vaccine strain on a clinical isolate from a TB patient, avoiding the use of laboratory-adapted strains such as H37Rv, Erdman, or CDC1551 . The cont system ,101,102.t system . PhoP ret ESAT-6 .pks2, pks3) involved in the biosynthesis of the polyketide-derived acyl trehalose  and sulfolipids. These components, besides their structural function, impede the recognition of Mtb by the immune system [fadD26, a gene of biosynthesis and the export of phthiocerol dimycocerosates (PDIM) which are major virulence-associated lipids of the MTB [Other significant virulence genes regulated by PhoP include genes of lipid metabolism (e.g., e system . The sec the MTB ,107. PDI the MTB ,109.MTBVAC has demonstrated that it is as safe as BCG in preclinical trials ,110. In Clinical trials of MTBVAC safety began in 2012, after 11 years of preclinical trials. Their result was that intradermal vaccination with MTBVAC was at least as safe as BCG and did not cause serious adverse events. Trials in healthy uninfected newborn infants also confirmed safety and greater immunogenicity compared with the same dose of BCG, suggesting a broader antigenic composition .–host interactions and the way this knowledge has translated into attempts to create new, more effective vaccines to prevent TB. To summarize, two widespread strategies for creating new TB vaccines have been discussed. The first is the introduction of genes of protective TB antigens and genes affecting antigen presentation. Strains of BCG and other less pathogenic mycobacteria-carrying genes encoding Mtb antigens have been actively developed. Additionally, the attenuated strain of Mtb, MTBVAC, which possesses the entire repertoire of TB antigens, and attenuating mutation different from BCG, is one of the most promising vaccines under clinical evaluation. The second way directs and shifts the interaction with the host cell, such as cell phagosome biology. For example, the integration of the genes providing the phagosome escape led to an increase in the intensity of the immune response. The most successful implementation of this approach has been embodied in a vaccine VPM1002 which is currently in phase III clinical trials.This article reviews the molecular mechanisms underlying MtbThe division into these two strategies is rather conditional since, in general, both are used in the development of vaccines. This seems to be the best way. However, it has been provided to highlight the areas for further improvement. Despite a huge amount of knowledge about BCG and lots of effort to make it perfect, several issues remain unsolved to date. A known disadvantage of BCG is the provoking BCGitises or severe generalized infections in some cases has to be noted. This drawback is present in recombinant BCG strains. Perhaps subunit vaccines solve this problem to a certain extent, but they cannot provide an organism with a wide repertoire of native antigens. Here, it will be worth mentioning one more time the positive nonspecific effects of BCG on immunity, including other diseases, which have already been discussed above. Obviously, subunit vaccines do not have similar properties, which undoubtedly makes them less favorable candidates.The ideal vaccine to replace BCG should balance the fine line between virulence and protection, providing a potent and long-term immune response to Mtb. The success of clinical trials on vaccines such as MTBVAC and VPM1002 appears to be very promising, but there is still a long road ahead. There is a large field for growth in both the above strategies. Identifying antigens associated with latency, as well as cryptic antigens, and amplifying the response to them, seems to be a promising step. The future directions aimed at bringing the creation of a new vaccine closer can be either in returning some antigens into weakened organisms/strains or in further attenuating the virulent ones. It is also necessary to improve our understanding of the action of immunomodulatory and virulent factors that allow Mtb to survive under the pressure of the immune system. A way of influencing these very pathways will permit the retrieval of a vaccine that activates the immune response in a more optimal way. It is not realized in nature due to the evolutionary mechanisms of Mtb.The combination of these approaches and the use of achievements of genetic engineering may lead to advanced recombinant live vaccines based on BCG, Mtb, or other mycobacteria that will allow the eradication of TB.