The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27kip1. This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications.

• MeSH
• Chloroform MeSH
• Lab-On-A-Chip Devices MeSH
• Microfluidics * methods   MeSH
• Microfluidic Analytical Techniques * MeSH
• Polymethyl Methacrylate chemistry   MeSH
• Publication type
• Journal Article MeSH

This paper introduces a complex novel concept and methodology for the creation of personalized biomedical appliances 3D-printed from certified biocompatible photopolymer resin Dental LT Clear (V2). The explained workflow includes intraoral and CT scanning, patient virtualization, digital appliance design, additive manufacturing, and clinical application with evaluation of the appliance intended for patients with cranio-facial syndromes. The presented concept defines virtual 3D fusion of intraoral optical scan and segmented CT as sufficient and accurate data defining the 3D surface of the face, intraoral and airway morphology necessary for the 3D design of complex personalized intraoral and extraoral parts of the orthopedic appliance. A central aspect of the concept is a feasible utilization of composite resin for biomedical prototyping of the sequence of marginally different appliances necessary to keep the pace with the patient rapid growth. Affordability, noninvasiveness, and practicality of the appliance update process shall be highlighted. The methodology is demonstrated on a particular case of two-year-old infant with Pierre Robin sequence. Materialization by additive manufacturing of this photopolymer provides a highly durable and resistant-to-fracture two-part appliance similar to a Tübingen palatal plate, for example. The paper concludes with the viability of the described method and material upon interdisciplinary clinical evaluation of experts from departments of orthodontics and cleft anomalies, pediatric pneumology and phthisiology, and pediatric otorhinolaryngology.

• Publication type
• Journal Article MeSH

Obrovskobuněčná myokarditida (OBM) je vzácné zánětlivé onemocnění srdce postihující často mladé osoby. Klinický průběh je typicky prudký, s fulminantním městnavým srdečním selháním. Prognóza je špatná, správná diagnóza je často stanovena až při pitvě. V této práci popisujeme případ této vzácné myokarditidy postihující 44letou, do té doby zdravou ženu, která byla hospitalizována na klinice anesteziologie a resuscitace po náhlé srdeční zástavě a mimonemocniční resuscitaci. Echokardiografie a zobrazovací vyšetření prokázala těžkou dysfunkci a dilataci obou komor, bez významného nálezu na koronárních tepnách. Dvanáct dní po přijetí pacientka zemřela pod obrazem městnavého srdečního selhání refrakterního na terapii. Při histologickém vyšetření srdce po pitvě byla v myokardu obou komor nalezena mnohočetná nekrotická ložiska s hustou zánětlivou celulizací s účastí hojných obrovských vícejaderných histiocytů, v souladu s diagnózou OBM. Článek diskutuje patologickou anatomii, patofyziologii a histologickou diferenciální diagnostiku této vzácné choroby, součásti je i přehled recentní literatury popisující neobvyklé a nové jednotky.

Giant cell myocarditis (GCM) is a rare inflammatory disease of the heart that often affects younger patients. The clinical course is typically rapid with fulminant congestive heart failure. Prognosis is poor; the proper diagnosis is often rendered at the autopsy. Herein, we present a prototypical case of this rare type of myocarditis, affecting a 44-year-old previously healthy woman who was referred to the intensive care department due to an acute onset cardiac arrest followed by resuscitation. The heart ultrasound and imaging examinations revealed a severe dysfunction and dilatation of both ventricles, without any significant finding in the coronary arteries. Twelve days after the initial presentation, the patient died due to congestive heart failure refractory to intensive therapy. The post-mortem histology of the heart revealed multiple small necrotic foci in the myocardium in both ventricles, with dense inflammatory infiltration with abundant multinucleated giant histiocytes, in line with a diagnosis of GCM. The natural history, pathophysiology, and histological differential diagnosis is discussed, together with review of the relevant literature including uncommon and emerging units.

• Keywords
• obrovskobuněčná myokarditida,
• MeSH
• Adult MeSH
• Echocardiography methods   MeSH
• Humans MeSH
• Myocardium pathology   MeSH
• Myocarditis * diagnosis   MeSH
• Autopsy MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Case Reports MeSH

Acute intoxication incidents due to neurotoxic organophosphate (OP) insecticides are occasionally reported, related either to suicidal attempts or occupational exposure due to the misuse of protective equipment. Among them, chlorpyrifos is a compound related to great controversy, which is still authorized and easily accessible in many countries around the world. However, to screen for its exposure markers, instrumental methods are commonly applied, which cannot enable rapid monitoring at an early stage of an intoxication. Therefore, in this study, a microfluidic paper-based analytical device (μPAD) able to rapidly screen for chlorpyrifos-oxon, the toxic chlorpyrifos metabolite, in human serum was developed and fully validated. The μPAD combines wax-printed butyrylcholinesterase (BChE) paper sensors, a lab-on-a-chip (LOC) prototype injector and a smartphone as the analytical detector. In principle, the wax-printed strips with adsorbed BChE are embedded into LOC injectors able to deliver samples and reagents on-demand. A smartphone reader was used to monitor the color development on the strips providing binary qualitative results. μPAD method performance characteristics were thoroughly evaluated in terms of specificity, detection capability (CCβ) and ruggedness. The developed analytical platform is rapid (results within 10 min), cost-efficient (0.70 €), potentially applicable at the point-of-need and attained a low CCβ (10 μg L-1 in human serum). Finally, μPAD characteristics were critically compared to well-established methods, namely an in-house BChE microplate assay and liquid chromatography tandem mass spectrometry.

• MeSH
• Smartphone MeSH
• Chlorpyrifos * MeSH
• Lab-On-A-Chip Devices MeSH
• Humans MeSH
• Microfluidics MeSH
• Microfluidic Analytical Techniques * MeSH
• Paper MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Poly(hydroxyalkanoates) are biodegradable and biocompatible polymers suitable for tissue engineering. Fused deposition modeling (FDM) belongs to modern rapid prototyping techniques for the fabrication of scaffolds. In this work, poly(3-hydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) were tested for FDM. Thermal and rheological properties of industrial PHAs were compared with poly(lactic acid) (PLA), which is a biodegradable polymer commonly used for FDM. The massive decrease in viscosity and loss of molecular weight of PHB and PHBV precluded their use for FDM. On the other hand, the thermal stability of PHBH was comparable to that of PLA. PHBH scaffolds prepared by FDM exhibited excellent mechanical properties, no cytotoxicity and large proliferation of mouse embryonic fibroblast cells within 96 h. The hydrolytic degradation of PHBH and PLA scaffolds tested in synthetic gastric juice for 52 days confirmed a faster degradation of PHBH than PLA. The decrease in molecular weight confirmed the first-order kinetics with a slightly higher (0.0169 day-1) degradation rate constant for PHBH as compared to the value (0.0107 day-1) obtained for PLA. These results indicate that PHBH could be used to produce scaffolds by FDM with application in tissue engineering.

• MeSH
• Biocompatible Materials chemistry   MeSH
• Caproates chemistry   MeSH
• 3-Hydroxybutyric Acid chemistry   MeSH
• Humans MeSH
• Mechanical Phenomena MeSH
• Molecular Structure MeSH
• Molecular Weight MeSH
• Mice MeSH
• Polymers chemistry   MeSH
• Rheology MeSH
• Temperature MeSH
• Thermogravimetry MeSH
• Tissue Scaffolds chemistry   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

BACKGROUND: Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. METHODOLOGY/PRINCIPAL FINDINGS: The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius. CONCLUSIONS/SIGNIFICANCE: We have successfully optimized the ICT, now also suitable to be used with whole canine blood. The test is able to reflect the seasonal fluctuation in dog exposure and showed a good detectability in a field population of naturally exposed dogs, particularly in areas with a high seroprevalence of bitten dogs. Furthermore, our study showed the existence of favorable cross-reactions with other sand fly vectors thereby expanding its use in the field.

• MeSH
• Insect Vectors parasitology   physiology   MeSH
• Immunoassay methods   MeSH
• Leishmania infantum physiology   MeSH
• Leishmaniasis blood   diagnosis   parasitology   veterinary   MeSH
• Mice, Inbred BALB C MeSH
• Dog Diseases blood   diagnosis   parasitology   MeSH
• Phlebotomus parasitology   physiology   MeSH
• Dogs MeSH
• Animals MeSH
• Check Tag
• Dogs MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: Identification of blood sources of hematophagous arthropods is crucial for understanding the transmission cycles of vector-borne diseases. Many different approaches towards host determination were proposed, including precipitin test, ELISA, DNA- and mass spectrometry-based methods; yet all face certain complications and limitations, mostly related to blood degradation. This study presents a novel method for blood meal identification, peptide mass mapping (PMM) analysis of host-specific hemoglobin peptides using MALDI-TOF mass spectrometry. METHODOLOGY/PRINCIPAL FINDINGS: To identify blood meal source, proteins from abdomens of engorged sand fly females were extracted, cleaved by trypsin and peptide fragments of host hemoglobin were sequenced using MALDI-TOF MS. The method provided correct host identification of 100% experimentally fed sand flies until 36h post blood meal (PBM) and for 80% samples even 48h PBM. In females fed on two hosts, both blood meal sources were correctly assigned for 60% of specimens until 36h PBM. In a validation study on field-collected females, the method yielded unambiguous host determination for 96% of specimens. The suitability of PMM-based MALDI-TOF MS was proven experimentally also on lab-reared Culex mosquitoes. CONCLUSIONS/SIGNIFICANCE: PMM-based MALDI-TOF MS analysis targeting host specific hemoglobin peptides represents a sensitive and cost-effective method with a fast and simple preparation protocol. As demonstrated here on phlebotomine sand flies and mosquitoes, it allows reliable and rapid blood source determination even 48h PBM with minimal material input and provides more robust and specific results than other currently used methods. This approach was also successfully tested on field-caught engorged females and proved to be a promising useful tool for large-scale screening of host preferences studies. Unlike other methods including MALDI-TOF protein profiling, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies.

• MeSH
• Blood Chemical Analysis methods   MeSH
• Species Specificity MeSH
• Hemoglobins chemistry   MeSH
• Humans MeSH
• Proof of Concept Study MeSH
• Peptides chemistry   MeSH
• Psychodidae chemistry   physiology   MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
• Feeding Behavior MeSH
• Tandem Mass Spectrometry MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH

Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).

• MeSH
• Bacterial Proteins isolation & purification   toxicity   MeSH
• Cytotoxins isolation & purification   toxicity   MeSH
• Detergents chemistry   MeSH
• Erythrocytes drug effects   MeSH
• Escherichia coli metabolism   MeSH
• Hemolysis MeSH
• Hemolysin Proteins isolation & purification   toxicity   MeSH
• Humans MeSH
• Lipopolysaccharides analysis   MeSH
• Urea chemistry   MeSH
• Cell Line, Tumor MeSH
• Octoxynol chemistry   MeSH
• Sheep MeSH
• THP-1 Cells MeSH
• Cell Survival drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Waste prevention and management become a significant issue worldwide to achieve sustainable development. Similar to many developing countries, Malaysia has faced severe problems in waste management due to its rapid economic growth and urbanisation. The municipal solid waste (MSW) production rate in Malaysia had increased significantly in a recent year, ranging from 0.8 to 1.25 kg/person∙d. The wastes generated contain a high amount of organic portion with high moisture content. Improper MSW management practice or delayed in waste collection and transportation can lead to severe health issues. This paper presents a case study in Johor Bahru, Malaysia (FOLO Farm), in which a composting prototype is used as the waste management technology to recycle the food and vegetable wastes. The greenhouse gases (GHG) mitigation and economic feasibility of the integrated composting and organic farming in this study are reported. This study showed a reduction of 27% of GHG by diverting the food and vegetable wastes from open dumping to the composting plant. Higher reduction rate (∼44%) can be achieved with better planning of waste collection route and applying the mitigation strategies during the composting process. By adapting the membership concept, this project not only ensures the economic feasibility of running a composting plant but also secures a channel for the growth of vegetable distribution. This study provides an insight into the feasibility and desirability to implement a pilot-scale composting for organic waste management to achieve the low carbon and self-sustain community.

• MeSH
• Organic Agriculture MeSH
• Composting * MeSH
• Waste Management * MeSH
• Refuse Disposal * MeSH
• Feasibility Studies MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Malaysia MeSH

Technologie vstřikování termoplastů je jednou z nejrozšířenějších technologií zpracování plastů například při výrobě automobilů, v elektrotechnice, elektronice, při výrobě spotřebního a sportovního zboží, v průmyslu obalů atd. Kniha se snaží komplexně popsat celý proces výroby, a to jak v teoretické, tak zejména v praktické rovině.