Association of 24-h central hemodynamics and stiffness with cardiovascular events and all-cause mortality. The VASOTENS Registry.

OBJECTIVES: In hemodialysis patients, central hemodynamics, stiffness, and wave reflections assessed through ambulatory blood pressure monitoring (ABPM) showed superior prognostic value for cardiovascular (CV) events than peripheral blood pressures (BPs). No such evidence is available for lower-risk hypertensive patients. METHODS: In 591 hypertensive patients (mean age 58 ±â€Š14 years, 49% males), ambulatory brachial and central BP, pulse wave velocity (PWV), and augmentation index (AIx) were obtained with a validated upper arm cuff-based pulse wave analysis technology. Information on treatment for hypertension (73% of patients), dyslipidemia (27%), diabetes (8%), CV disease history (25%), was collected. Patients were censored for CV events or all-cause death over 4.2 years. RESULTS: One hundred and four events (24 fatal) were recorded. Advanced age [hazard ratio and 95% confidence interval: 1.03 (1.01, 1.05), P = 0.0001], female sex [1.57 (1.05, 2.33), P = 0.027], CV disease [2.22 (1.50, 3.29), P = 0.0001], increased 24-h central pulse pressure (PP) [1.56 (1.05, 2.31), P = 0.027], PWV [1.59 (1.07, 2.36), P = 0.022], or AIx [1.59 (1.08, 2.36), P = 0.020] were significantly associated with a worse prognosis (univariate Cox regression analysis). The prognostic power of peripheral and central BPs was lower. However, PWV [1.02 (0.64, 1.63), P = 0.924], AIx [1.06 (0.66, 1.69), P = 0.823], and central PP [1.18 (0.76, 1.82), P = 0.471], were not significant predictors in multivariate analyses. CONCLUSIONS: In hypertensive patients, ambulatory central PP, PWV, and AIx are associated with an increased risk of CV morbidity and all-cause mortality. However, this association is not independent of other patient characteristics.

The role of the correlated motion(s) of the chromophore in photoswitching of green and red forms of the photoconvertible fluorescent protein mSAASoti.

Wild-type SAASoti and its monomeric variant mSAASoti can undergo phototransformations, including reversible photoswitching of the green form to a nonfluorescent state and irreversible green-to-red photoconversion. In this study, we extend the photochemistry of mSAASoti variants to enable reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues. In the case of mSAASoti it is M163T substitution that leads to the fastest switching and the most photostable variant, and reversible photoswitching can be observed for both green and red forms when expressed in eukaryotic cells. We obtained a 13-fold increase in the switching efficiency with the maximum switching contrast of the green form and the appearance of comparable switching of the red form for the C21N/M163T mSAASoti variant. The crystal structure of the C21N mSAASoti in its green on-state was obtained for the first time at 3.0 Å resolution, and it is in good agreement with previously calculated 3D-model. Dynamic network analysis reveals that efficient photoswitching occurs if motions of the 66H residue and phenyl fragment of chromophore are correlated and these moieties belong to the same community.

A pollen selection system links self and interspecific incompatibility in the Brassicaceae.

Self-incompatibility and recurrent transitions to self-compatibility have shaped the extant mating systems underlying the nonrandom mating critical for speciation in angiosperms. Linkage between self-incompatibility and speciation is illustrated by the shared pollen rejection pathway between self-incompatibility and interspecific unilateral incompatibility (UI) in the Brassicaceae. However, the pollen discrimination system that activates this shared pathway for heterospecific pollen rejection remains unknown. Here we show that Stigma UI3.1, the genetically identified stigma determinant of UI in Arabidopsis lyrata × Arabidopsis arenosa crosses, encodes the S-locus-related glycoprotein 1 (SLR1). Heterologous expression of A. lyrata or Capsella grandiflora SLR1 confers on some Arabidopsis thaliana accessions the ability to discriminate against heterospecific pollen. Acquisition of this ability also requires a functional S-locus receptor kinase (SRK), whose ligand-induced dimerization activates the self-pollen rejection pathway in the stigma. SLR1 interacts with SRK and interferes with SRK homomer formation. We propose a pollen discrimination system based on competition between basal or ligand-induced SLR1-SRK and SRK-SRK complex formation. The resulting SRK homomer levels would be sensed by the common pollen rejection pathway, allowing discrimination among conspecific self- and cross-pollen as well as heterospecific pollen. Our results establish a mechanistic link at the pollen recognition phase between self-incompatibility and interspecific incompatibility.

Multifunctionality of arginine residues in the active sites of non-canonical d-amino acid transaminases.

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical d-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical d-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical d-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of d-amino acids and α-keto acids, may help to tailor it for industrial applications.

Tridentate Nitrogen Ligand as a Tool for the Construction of Well-Defined Rare Earth Trichloride Complexes.

LnCl3(THF)3 (Ln = Y, La ÷ Nd, Sm ÷ Lu) readily react with the tridentate 1,3,5-trimethyl-1,3,5-triazacyclohexane (Me3tach) ligand to form mono- or binuclear lanthanide trichloride complexes, depending on the stoichiometry of the reaction and the ionic radius of the metal: mononuclear pseudosandwich [LnCl3(Me3tach)2], (Ln = Y, La ÷ Ho) or binuclear complexes [Ln2Cl6(Me3tach)3], or [LnCl3(Me3tach)(THF)]2 (Ln = Sm, Tb). Detailed analysis of the NMR data of [LnCl3(Me3tach)2] complexes with paramagnetic lanthanide ions showed that their structures remained unchanged in the toluene solution. A series of isomorphous complexes [LnCl3(Me3tach)(Py)2] (Ln = La, Sm, Tb, Er, Lu; Py = pyridine) have been obtained by the recrystallization of either mononuclear or binuclear complexes from pyridine. Complexes of terbium and europium ions with the Me3tach ligand exhibit relatively high quantum yields of metal-centered luminescence (0.39 and 0.32, respectively).

Hospital and long-term results of carotid endarterectomy in patients with different severity of coronary artery lesion according to syntax score.

AIM: Analysis of in-hospital and long-term results of carotid endarterectomy (CEE) in patients with different severity of coronary atherosclerosis. MATERIAL AND METHODS: This comparative, retrospective, open study for the period from January 2013 to April 2020 included 1719 patients operated on for occlusive-stenotic lesions of the internal carotid arteries (ICA). Classical and eversion CEA were used as revascularization strategies. The criteria for inclusion in the study were: 1. Presence of coronary angiography within six months before the present CEE; 2. A history of myocardial revascularization in patients with severe coronary lesions. Depending on the severity of coronary atherosclerosis, all patients were divided into 3 groups: Group 1-871 (50.7 %) patients - with the presence of hemodynamically significant stenosis of the coronary arteries (CA) with a history of myocardial revascularization; Group 2-496 (28.8 %) patients - with the presence of hemodynamically insignificant lesions of the coronary artery (up to 70 %, not inclusive, and the trunk of the left coronary artery, up to 50 %, not inclusive); Group 3-352 (20.5 %) patients - without signs of atherosclerotic lesions of the coronary artery. In group 1, the observation period was 56.8±23.2 months, in group 2-62.0±15.6 months, in group 3-58.1±20.4 months. RESULTS: During the hospital observation period, there were no significant intergroup differences in the number of complications. All cardiovascular events were detected in isolated cases. The most common injury was damage to the cranial nerves, diagnosed in every fifth patient in the total sample. The combined endpoint (CET), including death + myocardial infarction (MI) + acute cerebrovascular accident/transient ischemic attack (stroke/TIA), was 0.75 % (n=13). In the long-term follow-up period, when comparing survival curves, group 3 revealed the largest number of ischemic strokes (p = 0.007), myocardial infarction (p = 0.03), and CCT (p = 0.005). There were no intergroup differences in the number of deaths (p=0.62). CONCLUSION: The results of the study showed that there was no significant intergroup difference in the development of complications at the hospital postoperative stage. However, in the long-term follow-up period, a group of patients with isolated lesions of the ICA demonstrated a rapid increase in the number of MI, stroke/TIA, and a combined endpoint, which was apparently associated with low compliance and progression of atherosclerosis in previously unaffected arteries.

Structural peculiarities of tandem repeats and their clinical significance.

While it is well established that a mere 2% of human DNA nucleotides are involved in protein coding, the remainder of the DNA plays a vital role in the preservation of normal cellular genetic function. A significant proportion of tandem repeats (TRs) are present in non-coding DNA. TRs - specific sequences of nucleotides that entail numerous repetitions of a given fragment. In this study, we employed our novel algorithm grounded in finite automata theory, which we refer to as Dafna, to investigate for the first time the likelihood of these nucleotide sequences forming non-canonical DNA structures (NS). Such structures include G-quadruplexes, i-motifs, hairpins, and triplexes. The tandem repeats under consideration in our research encompassed sequences containing 1 to 6 nucleotides per repeated fragment. For comparison, we employed a set of randomly generated sequences of the same length (60 nucleotides) as a benchmark. The outcomes of our research exposed a disparity between the potential for NS formation in random sequences and tandem repeats. Our findings affirm that the propensity of DNA and RNA to form NS is closely tied to various genetic disorders, including Huntington's disease, Fragile X syndrome, and Friedreich's ataxia. In the concluding discussion, we present a proposal for a new therapeutic mechanism to address these diseases. This novel approach revolves around the ability of specific nucleic acid fragments to form multiple types of NS.

Characterization of two near-infrared genetically encoded voltage indicators.

Significance: Efforts starting more than 20 years ago led to increasingly well performing genetically encoded voltage indicators (GEVIs) for optical imaging at wavelengths <600 nm. Although optical imaging in the >600 nm wavelength range has many advantages over shorter wavelength approaches for mesoscopic in vivo monitoring of neuronal activity in the mammalian brain, the availability and evaluation of well performing near-infrared GEVIs are still limited. Aim: Here, we characterized two recent near-infrared GEVIs, Archon1 and nirButterfly, to support interested tool users in selecting a suitable near-infrared GEVI for their specific research question requirements. Approach: We characterized side-by-side the brightness, sensitivity, and kinetics of both near-infrared GEVIs in a setting focused on population imaging. Results: We found that nirButterfly shows seven-fold higher brightness than Archon1 under the same conditions and faster kinetics than Archon1 for population imaging without cellular resolution. But Archon1 showed larger signals than nirButterfly. Conclusions: Neither GEVI characterized here surpasses in all three key parameters (brightness, kinetics, and sensitivity), so there is no unequivocal preference for one of the two. Our side-by-side characterization presented here provides new information for future in vitro and ex vivo experimental designs.

Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from Blastococcus saxobsidens.

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine-a reversible inhibitor and analogue of (R)-1-phenylethylamine-a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the ß-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds.

Novel stable ytterbium acetylacetonate-quinaldinate complexes as single-molecule magnets and surprisingly efficient luminophores.

The first Yb complexes comprising a quinoline-2-carboxylate (quinaldinate, Q-) ligand, namely 1D-polymeric [Yb(acac)2(Q)]n (1, acac- is the acetylacetonate (pentane-2,4-dionate) anion) and mononuclear [Yb(acac)2(Q)(Phen)] (2, Phen is 1,10-phenanthroline), are reported. The bifunctionality of both complexes as field-induced single-molecule magnets (SMMs) and near IR luminophores has been revealed. The SMM properties of 1 and 2 have been discussed in terms of the geometry and composition of the coordination environment. Also, 1 is the first example of 1D-polymeric SMMs with the capped octahedral surrounding of Yb3+. The photoluminescence quantum yields (PLQYs) of 1 and 2 are 2 and 4%, respectively. The origins of this difference are discussed. Surprisingly, the PLQY value of 2 is high for compounds comprising a lot of C-H vibrational quenchers, being the highest one for reliably characterized Yb ß-diketonate complexes, and surpassing those for complexes with a broad range of anionic ligands. In this respect, the role of the Phen ligand is to tune the coordination mode of Q- thereby decreasing the energy of coordinating C-O oscillators rather than to act as a typical antenna ligand. These results can give rise to an alternative route to elaborate efficient Yb-based luminophores via the substitution of the ß-diketonate ligands controlled by the introduction of appropriate neutral ligands.

Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of five homologous subunits. The homopentameric α7-nAChR, abundantly expressed in the brain, is involved in the regulation of the neuronal plasticity and memory and undergoes phosphorylation by protein kinase A (PKA). Here, we extracted native α7-nAChR from murine brain, validated its assembly by cryo-EM and showed that phosphorylation by PKA in vitro enables its interaction with the abundant human brain protein 14-3-3ζ. Bioinformatic analysis narrowed the putative 14-3-3-binding site down to the fragment of the intracellular loop (ICL) containing Ser365 (Q361RRCSLASVEMS372), known to be phosphorylated in vivo. We reconstructed the 14-3-3ζ/ICL peptide complex and determined its structure by X-ray crystallography, which confirmed the Ser365 phosphorylation-dependent canonical recognition of the ICL by 14-3-3. A common mechanism of nAChRs' regulation by ICL phosphorylation and 14-3-3 binding that potentially affects nAChR activity, stoichiometry, and surface expression is suggested.

Remodeling the cellular stress response for enhanced genetic code expansion in mammalian cells.

Genetic code expansion (GCE) reprograms the translational machinery to site-specifically incorporate noncanonical amino acids (ncAAs) into a selected protein. The efficiency of GCE in mammalian cells might be compromised by cellular stress responses, among which, the protein kinase R(PKR)-dependent eIF2α phosphorylation pathway can reduce translation rates. Here we test several strategies to engineer the eIF2α pathway and boost the rate of translation and show that such interventions increase GCE efficiency in mammalian cells. In particular, addition of the N-terminal PKR fragment (1-174) provides a substantial enhancement in cytoplasmic GCE and also in GCE realized by OTOs (orthogonally translating designer organelles), which built on the principle of 2D phase separation to enable mRNA-selective ncAA incorporation. Our study demonstrates an approach for improving the efficiency of GCE and provides a means by which the power of designer organelles can be further optimized to tune protein translation.

Seroimmunotyping of African swine fever virus.

The extreme genetic and immunobiological heterogeneity exhibited by the African swine fever virus (ASFV) has been a significant impediment in the development of an efficacious vaccine against this disease. Consequently, the lack of internationally accepted protocols for the laboratory evaluation of candidate vaccines has become a major concern within the scientific community. The formulation of such protocols necessitates the establishment of a consensus at the international level on methods for the determination of homologous and heterologous isolates/strains of ASFV. The present article provides a comprehensive description of biological techniques employed in the classification of ASFV by seroimmunotypes. These techniques involve a holistic evaluation of ASFV isolates/strains based on their antigenic properties as determined by the hemadsorption inhibiting test (HAdI) using type-specific sera and an immunological test (IT) conducted on pigs inoculated with attenuated strains. The article outlines the methods for setting up the HAdI test, an IT on pigs, and the processes involved in the acquisition of type-specific serums for the HAdI test. It is pertinent to note that the definitive classification of seroimmunotype can only be ascertained after conducting an IT on pigs. The findings from the HAdI test or the phylogenetic analysis of the EP402R gene should be considered preliminary in nature.

In search for structural targets for engineering d-amino acid transaminase: modulation of pH optimum and substrate specificity.

The development of biocatalysts requires reorganization of the enzyme's active site to facilitate the productive binding of the target substrate and improve turnover number at desired conditions. Pyridoxal-5'-phosphate (PLP) - dependent transaminases are highly efficient biocatalysts for asymmetric amination of ketones and keto acids. However, transaminases, being stereoselective enzymes, have a narrow substrate specificity due to the ordered structure of the active site and work only in neutral-alkaline media. Here, we investigated the d-amino acid transaminase from Aminobacterium colombiense, with the active site organized differently from that of the canonical d-amino acid transaminase from Bacillus sp. YM-1. Using a combination of site-directed mutagenesis, kinetic analysis, molecular modeling, and structural analysis we determined the active site residues responsible for substrate binding, substrate differentiation, thermostability of a functional dimer, and affecting the pH optimum. We demonstrated that the high specificity toward d-glutamate/α-ketoglutarate is due to the interactions of a γ-carboxylate group with K237 residue, while binding of other substrates stems from the effectiveness of their accommodation in the active site optimized for d-glutamate/α-ketoglutarate binding. Furthermore, we showed that the K237A substitution shifts the catalytic activity optimum to acidic pH. Our findings are useful for achieving target substrate specificity and demonstrate the potential for developing and optimizing transaminases for various applications.

New class of RSO2-NHC ligands and Pd/RSO2-NHC complexes with tailored electronic properties and high performance in catalytic C-C and C-N bonds formation.

Imidazolium salts have found ubiquitous applications as N-heterocyclic carbene precursors and metal nanoparticle stabilizers in catalysis and metallodrug research. Substituents directly attached to the imidazole ring can have a significant influence on the electronic, steric, and other properties of NHC-proligands as well as their metal complexes. In the present study, for the first time, a new type of Pd/NHC complex with the RSO2 group directly attached to the imidazol-2-ylidene ligand core was designed and synthesized. The electronic properties as well as structural features of the new ligands were evaluated by means of experimental and computational methods. Interestingly, the introduction of a 4-aryl(alkyl)sulfonyl group only slightly decreased the electron donation, but it significantly increased the π-acceptance and slightly enhanced the buried volume (%Vbur) of new imidazol-2-ylidenes. New Pd/NHC complexes were obtained through selective C(2)H-palladation of some of the synthesized 4-RSO2-functionalized imidazolium salts under mild conditions. Several complexes demonstrated good activity in the catalysis of model cross-coupling reactions, outperforming the activity of similar complexes with non-substituted NHC ligands.

Preparation and Characterization of Hydrogel Films and Nanoparticles Based on Low-Esterified Pectin for Anticancer Applications.

Prospective adjuvant anticancer therapy development includes the establishing of drug delivery systems based on biocompatible and biodegradable carriers. We have designed films and nanoparticles (NPs) based on low-esterified pectin hydrogel using the ionic gelation method. We investigated morphology, nanomechanical properties, biocompatibility and anticancer activity. Hydrogel films are characterized by tunable viscoelastic properties and surface nanoarchitectonics through pectin concentration and esterification degree (DE), expressed in variable pore frequency and diameter. An in vitro study showed a significant reduction in metabolic activity and the proliferation of the U87MG human glioblastoma cell line, probably affected via the adhesion mechanism. Glioma cells formed neurosphere-like conglomerates with a small number of neurites when cultured on fully de-esterified pectin films and they did not produce neurites on the films prepared on 50% esterified pectin. Pectin NPs were examined in terms of size distribution and nanomechanical properties. The NPs' shapes were proved spherical with a mean diameter varying in the range of 90-115 nm, and a negative zeta potential from -8.30 to -7.86 mV, which indicated their stability. The NPs did not demonstrate toxic effect on cells or metabolism inhibition, indicating good biocompatibility. Nanostructured biomaterials prepared on low-esterified pectins could be of interest for biomedical applications in adjuvant anticancer therapy and for designing drug delivery systems.

How to Develop Drug Delivery System Based on Carbohydrate Nanoparticles Targeted to Brain Tumors.

Brain tumors are the most difficult to treat, not only because of the variety of their forms and the small number of effective chemotherapeutic agents capable of suppressing tumor cells, but also limited by poor drug transport across the blood-brain barrier (BBB). Nanoparticles are promising drug delivery solutions promoted by the expansion of nanotechnology, emerging in the creation and practical use of materials in the range from 1 to 500 nm. Carbohydrate-based nanoparticles is a unique platform for active molecular transport and targeted drug delivery, providing biocompatibility, biodegradability, and a reduction in toxic side effects. However, the design and fabrication of biopolymer colloidal nanomaterials have been and remain highly challenging to date. Our review is devoted to the description of carbohydrate nanoparticle synthesis and modification, with a brief overview of the biological and promising clinical outcomes. We also expect this manuscript to highlight the great potential of carbohydrate nanocarriers for drug delivery and targeted treatment of gliomas of various grades and glioblastomas, as the most aggressive of brain tumors.

Synthesis of substituted 8H-benzo[h]pyrano[2,3-f]quinazolin-8-ones via photochemical 6π-electrocyclization of pyrimidines containing an allomaltol fragment.

For the first time, we elaborated a method for the synthesis of pyrimidines containing an allomaltol unit. The suggested approach is based on the reaction of 2-(1-(dimethylamino)-3-oxo-3-arylprop-1-en-2-yl)-3-hydroxy-6-methyl-4H-pyran-4-ones with cyanamide. The photochemical behavior of the obtained pyrimidines was investigated. It was shown that for the hydroxy derivatives the main pathway of phototransformation is a 6π-electrocyclization of the 1,3,5-hexatriene system and subsequent [1,9]-H sigmatropic shift leading to dihydrobenzo[h]pyrano[2,3-f]quinazolines. At the same time, for methylated analogues the photoreaction proceeds in two directions resulting in the formation of a mixture of the corresponding dihydrobenzo[h]pyrano[2,3-f]quinazolines and polyaromatic products. The obtained dihydro derivatives are stable compounds and do not undergo aromatization upon further UV irradiation. The structures of two of the dihydrobenzo[h]pyrano[2,3-f]quinazolines were confirmed by X-ray diffraction analysis. Based on the performed studies, a two-stage telescopic method for the synthesis of polyaromatic benzo[h]pyrano[2,3-f]quinazolines including the initial photocyclization of the starting pyrimidines and the final dehydration was proposed.

Subsequent Immunization of Pigs with African Swine Fever Virus (ASFV) Seroimmunotype IV Vaccine Strain FK-32/135 and by Recombinant Plasmid DNA Containing the CD2v Derived from MK-200 ASFV Seroimmunotype III Strain Does Not Protect from Challenge with ASFV Seroimmunotype III.

Understanding the immunological mechanisms of protection and the viral proteins involved in the induction of a protective immune response to the African swine fever virus (ASFV) is still limited. In the last years, the CD2v protein (gp110-140) of the ASFV has been proven to be a serotype-specific protein. Current work is devoted to the investigation of the possibility of creating protection against virulent ASFV strain Mozambique-78 (seroimmunotype III) in pigs previously vaccinated with vaccine strain FK-32/135 (seroimmunotype IV) and then immunized with the pUBB76A_CD2v plasmid, containing a chimeric nucleotide sequence from the CD2v protein gene (EP402R, nucleotides from 49 to 651) from the MK-200 strain (seroimmunotype III). Vaccination with the ASFV vaccine strain FK-32/135 protects pigs from the disease caused by the strain with homologous seroimmunotype-France-32 (seroimmunotype IV). Our attempt to create balanced protection against virulent strain Mozambique-78 (seroimmunotype III) by induction of both humoral factors of immunity (by vaccination with strain FK-32/135 of seroimmunotype IV) and serotype-specific cellular immunity (by immunization with the plasmid pUBB76A_CD2v of seroimmunotype III) was unsuccessful.

Artificial confocal microscopy for deep label-free imaging.

Widefield microscopy of optically thick specimens typically features reduced contrast due to "spatial crosstalk", in which the signal at each point in the field of view is the result of a superposition from neighbouring points that are simultaneously illuminated. In 1955, Marvin Minsky proposed confocal microscopy as a solution to this problem. Today, laser scanning confocal fluorescence microscopy is broadly used due to its high depth resolution and sensitivity, but comes at the price of photobleaching, chemical, and photo-toxicity. Here, we present artificial confocal microscopy (ACM) to achieve confocal-level depth sectioning, sensitivity, and chemical specificity, on unlabeled specimens, nondestructively. We equipped a commercial laser scanning confocal instrument with a quantitative phase imaging module, which provides optical path-length maps of the specimen in the same field of view as the fluorescence channel. Using pairs of phase and fluorescence images, we trained a convolution neural network to translate the former into the latter. The training to infer a new tag is very practical as the input and ground truth data are intrinsically registered, and the data acquisition is automated. The ACM images present significantly stronger depth sectioning than the input (phase) images, enabling us to recover confocal-like tomographic volumes of microspheres, hippocampal neurons in culture, and 3D liver cancer spheroids. By training on nucleus-specific tags, ACM allows for segmenting individual nuclei within dense spheroids for both cell counting and volume measurements. In summary, ACM can provide quantitative, dynamic data, nondestructively from thick samples, while chemical specificity is recovered computationally.