In vivo Validation of Hsp90 Trans-splicing in Giardia lamblia: Highlighting the Role of Cis-elements.

Giardia lambliacauses giardiasis, one of the most common human infectious diseases globally. Previous studies from our lab have shown that hsp90 gene ofGiardia is split into two halves, namely hspN and hspC. The independent pre-mRNAs of these split genes join by trans-splicing, producing a full-length Hsp90 (FlHsp90) mRNA. Genetic manipulation of the participating genes is necessary to understand the mechanism and significance of such trans-splicing based expression of Hsp90. In this study, we have performed transfection based exogenous expression of hspN and/or hspC in G. lamblia. We electroporated a plasmid containing the Avi-tagged hspN component of Hsp90 and examined its fate in G. lamblia. We show that the exogenously expressed hspN RNA gets trans-spliced to endogenously expressed hspC RNA, giving rise to a hybrid-FlHsp90. We highlight the importance of cis-elements in this trans-splicing reaction through mutational analysis. The episomal plasmid carrying deletions in the intronic region of hspN, showed inhibition of the trans-splicing reaction.Additionally, exogenous hspC RNA also followed the same fate as of exogenous hspN, while upon co-transfection with episomal hspN, they underwent trans-splicing with each other. Using eGFP as a test protein, we have shown that intronic sequences of hsp90 gene can guide trans-splicing mediated repair of any associated exonic sequences. Our study provides in vivo validation of Hsp90 trans-splicing, showing crucial role of cis-elements and importantly highlights the potential of hsp90 intronic sequences to function as a minimal splicing tool.

Adaptation of the late ISC pathway in the anaerobic mitochondrial organelles of Giardia intestinalis.

Mitochondrial metabolism is entirely dependent on the biosynthesis of the [4Fe-4S] clusters, which are part of the subunits of the respiratory chain. The mitochondrial late ISC pathway mediates the formation of these clusters from simpler [2Fe-2S] molecules and transfers them to client proteins. Here, we characterized the late ISC pathway in one of the simplest mitochondria, mitosomes, of the anaerobic protist Giardia intestinalis that lost the respiratory chain and other hallmarks of mitochondria. In addition to IscA2, Nfu1 and Grx5 we identified a novel BolA1 homologue in G. intestinalis mitosomes. It specifically interacts with Grx5 and according to the high-affinity pulldown also with other core mitosomal components. Using CRISPR/Cas9 we were able to establish full bolA1 knock out, the first cell line lacking a mitosomal protein. Despite the ISC pathway being the only metabolic role of the mitosome no significant changes in the mitosome biology could be observed as neither the number of the mitosomes or their capability to form [2Fe-2S] clusters in vitro was affected. We failed to identify natural client proteins that would require the [2Fe-2S] or [4Fe-4S] cluster within the mitosomes, with the exception of [2Fe-2S] ferredoxin, which is itself part of the ISC pathway. The overall uptake of iron into the cellular proteins remained unchanged as also observed for the grx5 knock out cell line. The pull-downs of all late ISC components were used to build the interactome of the pathway showing specific position of IscA2 due to its interaction with the outer mitosomal membrane proteins. Finally, the comparative analysis across Metamonada species suggested that the adaptation of the late ISC pathway identified in G. intestinalis occurred early in the evolution of this supergroup of eukaryotes.

Two domains of Tim50 coordinate translocation of proteins across the two mitochondrial membranes.

Hundreds of mitochondrial proteins with N-terminal presequences are translocated across the outer and inner mitochondrial membranes via the TOM and TIM23 complexes, respectively. How translocation of proteins across two mitochondrial membranes is coordinated is largely unknown. Here, we show that the two domains of Tim50 in the intermembrane space, named core and PBD, both have essential roles in this process. Building upon the surprising observation that the two domains of Tim50 can complement each other in trans, we establish that the core domain contains the main presequence-binding site and serves as the main recruitment point to the TIM23 complex. On the other hand, the PBD plays, directly or indirectly, a critical role in cooperation of the TOM and TIM23 complexes and supports the receptor function of Tim50. Thus, the two domains of Tim50 both have essential but distinct roles and together coordinate translocation of proteins across two mitochondrial membranes.

The Global Otolaryngology-Head and Neck Surgery Workforce.

Importance: A core component of delivering care of head and neck diseases is an adequate workforce. The World Health Organization report, Multi-Country Assessment of National Capacity to Provide Hearing Care, captured primary workforce estimates from 68 member states in 2012, noting that response rates were a limitation and that updated more comprehensive data are needed. Objective: To establish comprehensive workforce metrics for global otolaryngology-head and neck surgery (OHNS) with updated data from more countries/territories. Design, Setting, and Participants: A cross-sectional electronic survey characterizing the OHNS workforce was disseminated from February 10 to June 22, 2022, to professional society leaders, medical licensing boards, public health officials, and practicing OHNS clinicians. Main Outcome: The OHNS workforce per capita, stratified by income and region. Results: Responses were collected from 121 of 195 countries/territories (62%). Survey responses specifically reported on OHNS workforce from 114 countries/territories representing 84% of the world's population. The global OHNS clinician density was 2.19 (range, 0-61.7) OHNS clinicians per 100 000 population. The OHNS clinician density varied by World Bank income group with higher-income countries associated with a higher density of clinicians. Regionally, Europe had the highest clinician density (5.70 clinicians per 100 000 population) whereas Africa (0.18 clinicians per 100 000 population) and Southeast Asia (1.12 clinicians per 100 000 population) had the lowest. The OHNS clinicians deliver most of the surgical management of ear diseases and hearing care, rhinologic and sinus diseases, laryngeal disorders, and upper aerodigestive mucosal cancer globally. Conclusion and Relevance: This cross-sectional survey study provides a comprehensive assessment of the global OHNS workforce. These results can guide focused investment in training and policy development to address disparities in the availability of OHNS clinicians.

Remote psychophysical testing of smell in patients with persistent olfactory dysfunction after COVID-19.

Olfactory dysfunction associated with coronavirus 2 (SARS-CoV-2) infection is in most cases transient, recovering spontaneously within a few days. However, in some patients it persists for a long time, affects their everyday life and endangers their health. Hence, we focused on patients with persistent loss of smell. The aim of this study was to evaluate olfactory dysfunction using a standardized test. Due to the pandemic, olfactory testing was performed online. Smell tests (Odorized Markers Test, OMT) were sent home to the patients. Together with the smell self-testing, participants reported and assessed several parameters (age, sex, subjective assessment of smell and taste, nasal patency, etc.) in an online questionnaire. Based on the questionnaire outcomes, the results were sent to the patients along with a list of participating otolaryngologists who provided them with professional care. From March to June 2021, 1025 patients requested smell testing, of these, 824 met the inclusion criteria of this study. The duration of the olfactory dysfunction at the time of testing ranged from 1 month to 1 year. Using the OMT, impaired smell ability-anosmia or hyposmia-was confirmed in 82.6% of participants. A total of 17.4% of participants were determined to be normosmic however, more than 50% of them complained of parosmia and/or phantosmia. Our study demonstrates the relevance of psychophysical smell testing and its suitability for remote use during the pandemic. This study also revealed several correlations between prolonged olfactory dysfunction and the monitored parameters.

Bacterial Type II Secretion System and Its Mitochondrial Counterpart.

Over the billions of years that bacteria have been around, they have evolved several sophisticated protein secretion nanomachines to deliver toxins, hydrolytic enzymes, and effector proteins into their environments. Of these, the type II secretion system (T2SS) is used by Gram-negative bacteria to export a wide range of folded proteins from the periplasm across the outer membrane. Recent findings have demonstrated that components of the T2SS are localized in mitochondria of some eukaryotic lineages, and their behavior is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). This review focuses on recent advances in the field and discusses open questions concerning the function and evolution of miT2SSs.

Unilateral macrocystic dysplasia and contralateral agenesis in a monoamniotic twin.

OBJECTIVE: We present a case report of a congenital malformation of the uropoetic tract in one of the monoamniotic twins. CASE REPORT: A 24-year-old primigravida with male monochorionic monoamniotic twins was dia-gnosed with congenital malformation in fetus A at 24 weeks of gestation. Ultrasound verified macrocystic dysplasia and contralateral renal agenesis. Planned caesarean section was performed after the observational management of the patient in the 34th gestational week. In fetus B, a physiological finding was confirmed on the postpartum ultrasonography. In fetus A, CT examination of the abdomen confirmed the finding of left kidney agenesis and polycystic degeneration of the right kidney. Exitus letalis was stated on the newborns 5th day. CONCLUSION: The occurrence of the described combination of congenital malformation in monoamniotic twins is rare. When dysplasia significantly affects the function of the parenchyma, renal agenesis with multicystic dysplasia of the other kidney is a condition incompatible with life. For the intrauterine survival of the affected fetus, the normal renal function of the twin was important and thus the normal volume of amniotic fluid was maintained. As a result, the fetus did not develop extrarenal symptoms of the Potter sequence in the described case - especially pulmonary hypoplasia and the newborn was able to ventilate spontaneously. The death was caused by the consequences of renal failure associated with anuria.

Structurally derived universal mechanism for the catalytic cycle of the tail-anchored targeting factor Get3.

Tail-anchored (TA) membrane proteins, accounting for roughly 2% of proteomes, are primarily targeted posttranslationally to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. Here, we identify the GET pathway in Giardia intestinalis and present the structure of the Get3-client complex in the critical postnucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work resolves key conundrums and allows for a complete model of the dramatic conformational landscape of Get3.

Covert postpartum urinary retention: causes and consequences (PAREZ study).

INTRODUCTION AND HYPOTHESIS: Increased post-voiding residual volume (PVRV), known as covert postpartum urinary retention (PUR), is an asymptomatic condition with unknown long-term adverse effects. The objectives were to determine the frequency of this phenomenon 3 days after delivery and to examine the associated risk factors and consequences of the increased residuum on women´s health 6 weeks postpartum. METHODS: We carried out a prospective observational study including a total of 926 primiparous women, giving birth to singletons. All participants underwent ultrasound determination of PVRV on day 3 postpartum. Then, risk factors were determined using logistic regression analysis. After 6 weeks, participants were invited to return for PVRV determination and to complete urogynecological and general health questionnaires. Using these data, the consequences of increased PVRV were determined. RESULTS: A total of n=90 women were diagnosed with abnormal PVRV. Mean age in the studied population was 30.4 years, BMI prior to delivery 27.8, weight of the newborn 3,420 g, and percentage of cesarean sections 15.9%. Gestational week (p=0.043), vaginal tear (p=0.032), and induction of labor (p=0.003) were risk factors for covert PUR. Puerperal incidence of urinary tract infection was 1.1% (6 out of 526) and of urinary incontinence 29.2% (155 out of 530), with no differences between the groups. In the second examination, covert PUR was no longer present, and the values of residual urine decreased for all patients in the case group. No statistically significant differences were observed in questionnaire scores in general health and wellbeing perceptions between the groups. CONCLUSIONS: We have found a few significant obstetrical-pediatric risk factors for abnormal PVRVs. Data from the follow-up suggest that covert PUR has no impact on morbidity and quality of life 6 weeks postpartum. Therefore, abnormal PVRV is a self-limited phenomenon with a tendency toward self-correction. Our findings support those of previous studies that advocate against screening for asymptomatic retention in the postpartum period, despite some similar previous recommendations.

Efficient CRISPR/Cas9-mediated gene disruption in the tetraploid protist Giardia intestinalis.

CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.

Inheritance of the reduced mitochondria of Giardia intestinalis is coupled to the flagellar maturation cycle.

BACKGROUND: The presence of mitochondria is a distinguishing feature between prokaryotic and eukaryotic cells. It is currently accepted that the evolutionary origin of mitochondria coincided with the formation of eukaryotes and from that point control of mitochondrial inheritance was required. Yet, the way the mitochondrial presence has been maintained throughout the eukaryotic cell cycle remains a matter of study. Eukaryotes control mitochondrial inheritance mainly due to the presence of the genetic component; still only little is known about the segregation of mitochondria to daughter cells during cell division. Additionally, anaerobic eukaryotic microbes evolved a variety of genomeless mitochondria-related organelles (MROs), which could be theoretically assembled de novo, providing a distinct mechanistic basis for maintenance of stable mitochondrial numbers. Here, we approach this problem by studying the structure and inheritance of the protist Giardia intestinalis MROs known as mitosomes. RESULTS: We combined 2D stimulated emission depletion (STED) microscopy and focused ion beam scanning electron microscopy (FIB/SEM) to show that mitosomes exhibit internal segmentation and conserved asymmetric structure. From a total of about forty mitosomes, a small, privileged population is harnessed to the flagellar apparatus, and their life cycle is coordinated with the maturation cycle of G. intestinalis flagella. The orchestration of mitosomal inheritance with the flagellar maturation cycle is mediated by a microtubular connecting fiber, which physically links the privileged mitosomes to both axonemes of the oldest flagella pair and guarantees faithful segregation of the mitosomes into the daughter cells. CONCLUSION: Inheritance of privileged Giardia mitosomes is coupled to the flagellar maturation cycle. We propose that the flagellar system controls segregation of mitochondrial organelles also in other members of this supergroup (Metamonada) of eukaryotes and perhaps reflects the original strategy of early eukaryotic cells to maintain this key organelle before mitochondrial fusion-fission dynamics cycle as observed in Metazoa was established.

Fates of Sec, Tat, and YidC Translocases in Mitochondria and Other Eukaryotic Compartments.

Formation of mitochondria by the conversion of a bacterial endosymbiont was a key moment in the evolution of eukaryotes. It was made possible by outsourcing the endosymbiont's genetic control to the host nucleus, while developing the import machinery for proteins synthesized on cytosolic ribosomes. The original protein export machines of the nascent organelle remained to be repurposed or were completely abandoned. This review follows the evolutionary fates of three prokaryotic inner membrane translocases Sec, Tat, and YidC. Homologs of all three translocases can still be found in current mitochondria, but with different importance for mitochondrial function. Although the mitochondrial YidC homolog, Oxa1, became an omnipresent independent insertase, the other two remained only sporadically present in mitochondria. Only a single substrate is known for the mitochondrial Tat and no function has yet been assigned for the mitochondrial Sec. Finally, this review compares these ancestral mitochondrial proteins with their paralogs operating in the plastids and the endomembrane system.

Microstructural Characterization of Laser Weld of Hot-Stamped Al-Si Coated 22MnB5 and Modification of Weld Properties by Hybrid Welding.

The presence of Al-Si coating on 22MnB5 leads to the formation of large ferritic bands in the dominantly martensitic microstructure of butt laser welds. Rapid cooling of laser weld metal is responsible for insufficient diffusion of coating elements into the steel and incomplete homogenization of weld fusion zone. The Al-rich regions promote the formation of ferritic solid solution. Soft ferritic bands cause weld joint weakening. Laser welds reached only 64% of base metal's ultimate tensile strength, and they always fractured in the fusion zone during the tensile tests. We implemented hybrid laser-TIG welding technology to reduce weld cooling rate by the addition of heat of the arc. The effect of arc current on weld microstructure and mechanical properties was investigated. Thanks to the slower cooling, the large ferritic bands were eliminated. The hybrid welds reached greater ultimate tensile strength compared to laser welds. The location of the fracture moved from the fusion zone to a tempered heat-affected zone characterized by a drop in microhardness. The minimum of microhardness was independent of heat input in this region.

Analysis of diverse eukaryotes suggests the existence of an ancestral mitochondrial apparatus derived from the bacterial type II secretion system.

The type 2 secretion system (T2SS) is present in some Gram-negative eubacteria and used to secrete proteins across the outer membrane. Here we report that certain representative heteroloboseans, jakobids, malawimonads and hemimastigotes unexpectedly possess homologues of core T2SS components. We show that at least some of them are present in mitochondria, and their behaviour in biochemical assays is consistent with the presence of a mitochondrial T2SS-derived system (miT2SS). We additionally identified 23 protein families co-occurring with miT2SS in eukaryotes. Seven of these proteins could be directly linked to the core miT2SS by functional data and/or sequence features, whereas others may represent different parts of a broader functional pathway, possibly also involving the peroxisome. Its distribution in eukaryotes and phylogenetic evidence together indicate that the miT2SS-centred pathway is an ancestral eukaryotic trait. Our findings thus have direct implications for the functional properties of the early mitochondrion.

Retortamonads from vertebrate hosts share features of anaerobic metabolism and pre-adaptations to parasitism with diplomonads.

Although the mitochondria of extant eukaryotes share a single origin, functionally these organelles diversified to a great extent, reflecting lifestyles of the organisms that host them. In anaerobic protists of the group Metamonada, mitochondria are present in reduced forms (also termed hydrogenosomes or mitosomes) and a complete loss of mitochondrion in Monocercomonoides exilis (Metamonada:Preaxostyla) has also been reported. Within metamonads, retortamonads from the gastrointestinal tract of vertebrates form a sister group to parasitic diplomonads (e.g. Giardia and Spironucleus) and have also been hypothesized to completely lack mitochondria. We obtained transcriptomic data from Retortamonas dobelli and R. caviae and searched for enzymes of the core metabolism as well as mitochondrion- and parasitism-related proteins. Our results indicate that retortamonads have a streamlined metabolism lacking pathways for metabolites they are probably capable of obtaining from prey bacteria or their environment, reminiscent of the biochemical arrangement in other metamonads. Retortamonads were surprisingly found do encode homologs of components of Giardia's remarkable ventral disk, as well as homologs of regulatory NEK kinases and secreted lytic enzymes known for involvement in host colonization by Giardia. These can be considered pre-adaptations of these intestinal microorganisms to parasitism. Furthermore, we found traces of the mitochondrial metabolism represented by iron­sulfur cluster assembly subunits, subunits of mitochondrial translocation and chaperone machinery and, importantly, [FeFe]­hydrogenases and hydrogenase maturases (HydE, HydF and HydG). Altogether, our results strongly suggest that a remnant mitochondrion is still present.

The risk of urine bacterial colonisation in patients with a permanent catheter after caesarean section.

We present an observational study, conducted in Slovakia, concerning the occurrence of newly acquired urine colonisations in women with Foley catheters after a Caesarean section. A sample of urine was taken from each patient when the Foley catheter was first inserted, before the operation and was sent to the lab for culture. Later, a sample of urine was taken during the removal of the Foley catheter. Out of 176 women, the second urine sample culture result was positive in 13 women. Of those nine women had a positive pathogenic strain (5.1%). The prevalence of asymptomatic bacteriuria in our cohort was 7.7%. De novo acquired colonisation of urine was confirmed in 5.1% of cases. The only confirmed risk factor was delivery by an acute Caesarean section.Impact statementWhat is already known on this subject?: It is well known that catheterisation increases risk of colonisation of lower urinary tract by pathogens. However, the extent of this risk is not determined because there are no studies of de novo colonisation in women with sterile urine before catheterisation. According to literature approximately 8% of women have asymptomatic bacteriuria, which could be confounding factor in previous studies.What do the results of this study add?: Our study excluded women with positive bacteriuria before insertion of Foley catheter. Therefore, the study only assesses de novo colonisation, dependent on insertion of Foley catheter during caesarean section.What are the implications of these findings for clinical practice and/or further research?: De novo colonisation was observed in 5.1% of women in our cohort, with emergency caesarean section as a confirmed risk factor. Therefore, practitioners should consider avoiding catheterisation during caesarean section.

Impact of implementing a multifaceted intervention to reduce rates of cesarean section: A quality-improvement study.

OBJECTIVE: To assess the impact of a multifaceted intervention on reducing the rate of cesarean section (CS) without negatively affecting the rate of perinatal mortality. METHODS: A retrospective analysis of CS was performed before and after the implementation of a quality-improvement (QI) intervention in a university-affiliated teaching hospital in the Slovak Republic. All women who gave birth in 2015 (pre-intervention) and 2018 (post-intervention) were included. The different components of the intervention were introduced from September 2016. The main outcome was the overall rate of CS. A subanalysis by Robson groups was undertaken. RESULTS: After the implementation of the QI intervention, there was a 33.5% reduction in the rate of CS compared to the pre-intervention period where the rate reduced from 33.7% to 22.4% (P<0.001; relative risk 0.66, 95% confidence interval [CI] 0.61-0.72; Number Needed to Treat (NNT) 8.8, 95% CI 7.3-11.2). The main contributors were the reduction in elective CS for non-obstetric reasons, a reduction in intrapartum CS for failure to progress, and an increase in the number of vaginal births after CS in the post-intervention period. CONCLUSION: The implementation of the composite QI intervention led to a significant reduction in the rate of CS without affecting the rate of perinatal mortality.

The evolution of the Puf superfamily of proteins across the tree of eukaryotes.

BACKGROUND: Eukaryotic gene expression is controlled by a number of RNA-binding proteins (RBP), such as the proteins from the Puf (Pumilio and FBF) superfamily (PufSF). These proteins bind to RNA via multiple Puf repeat domains, each of which specifically recognizes a single RNA base. Recently, three diversified PufSF proteins have been described in model organisms, each of which is responsible for the maturation of ribosomal RNA or the translational regulation of mRNAs; however, less is known about the role of these proteins across eukaryotic diversity. RESULTS: Here, we investigated the distribution and function of PufSF RBPs in the tree of eukaryotes. We determined that the following PufSF proteins are universally conserved across eukaryotes and can be broadly classified into three groups: (i) Nop9 orthologues, which participate in the nucleolar processing of immature 18S rRNA; (ii) 'classical' Pufs, which control the translation of mRNA; and (iii) PUM3 orthologues, which are involved in the maturation of 7S rRNA. In nearly all eukaryotes, the rRNA maturation proteins, Nop9 and PUM3, are retained as a single copy, while mRNA effectors ('classical' Pufs) underwent multiple lineage-specific expansions. We propose that the variation in number of 'classical' Pufs relates to the size of the transcriptome and thus the potential mRNA targets. We further distinguished full set of PufSF proteins in divergent metamonad Giardia intestinalis and initiated their cellular and biochemical characterization. CONCLUSIONS: Our data suggest that the last eukaryotic common ancestor (LECA) already contained all three types of PufSF proteins and that 'classical' Pufs then underwent lineage-specific expansions.

Mitochondrial dynamics in parasitic protists.

The shape and number of mitochondria respond to the metabolic needs during the cell cycle of the eukaryotic cell. In the best-studied model systems of animals and fungi, the cells contain many mitochondria, each carrying its own nucleoid. The organelles, however, mostly exist as a dynamic network, which undergoes constant cycles of division and fusion. These mitochondrial dynamics are driven by intricate protein machineries centered around dynamin-related proteins (DRPs). Here, we review recent advances on the dynamics of mitochondria and mitochondrion-related organelles (MROs) of parasitic protists. In contrast to animals and fungi, many parasitic protists from groups of Apicomplexa or Kinetoplastida carry only a single mitochondrion with a single nucleoid. In these groups, mitochondrial division is strictly coupled to the cell cycle, and the morphology of the organelle responds to the cell differentiation during the parasite life cycle. On the other hand, anaerobic parasitic protists such as Giardia, Entamoeba, and Trichomonas contain multiple MROs that have lost their organellar genomes. We discuss the function of DRPs, the occurrence of mitochondrial fusion, and mitophagy in the parasitic protists from the perspective of eukaryote evolution.

In vitro testing of flash-frozen sublingual membranes for storage and reproducible permeability studies of macromolecular drugs from solution or nanofiber mats.

Sublingual drug delivery allows systemic delivery of drug without difficulties connected with the gastrointestinal pathway. We developed a new simple protocol for easy-to-use processing and storage of porcine sublingual mucosal membrane for in vitro studies using "flash freezing" in liquid nitrogen. All the dextrans used as mucosal membrane integrity and permeability markers permeated only slowly through sublingual mucosa illustrating usability both the "fresh" and "flash frozen" sublingual membranes whereas conventional cold storage "frozen" membranes have shown significantly higher permeabilities for macromolecules due to the sustained damage. The permeability values were too low to expect dextrans to be potential carriers at this context. To test albumin as a drug carrier we compared FITC-albumin permeation from solutions vs. nanofiber mats donors. To increase the amounts and prolong the transport, we manufactured nanofiber mats loaded with fluorescently marked albumin using well-scalable electrospinning technology. Nanofiber mats have allowed albumin passage through the sublingual membrane in similar amounts as from the pure artificial saliva solution. Since salivary washout strictly limits the duration of liquid dosages, nanofiber mats may thus permit prolonged sublingual administration.