[Surgical technique of keratoplasty with simultaneous implantation of a continuous ring or ring segment into the graft]. / Khirurgicheskaya tekhnika keratoplastiki s odnomomentnoi implantatsiei tsel'nogo ili razomknutogo kol'tsa v transplantat.

The article presents the surgical technique of penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK) with femtosecond-laser assistance involving simultaneous implantation of an intracorneal continuous ring (ICCR) or an intracorneal ring segment (ICRS) into the graft. Surgical technique no. 1 - keratoplasty with simultaneous implantation of ICRS. Intrastromal circular tunnel is formed in the central zone of donor cornea using femtosecond laser. Then penetrating trepanation 8.1 mm in diameter is performed symmetrically to the formed tunnel. After preparing penetrating or lamellar recipient bed, suture fixation is placed in the corneal transplant, then the ICRS is implanted into the graft. Surgical technique no. 2 - keratoplasty with simultaneous implantation of ICCR. The donor cornea is dissected from periphery to center using femtosecond laser. Central zone remains untouched. A large diameter full-thickness trepanation is performed and the donor cornea is divided into the anterior and posterior layers. The ICCR is put on the donor cornea while holding the posterior layer with forceps. Penetrating or lamellar recipient bed is prepared, then the corneal graft is fixed with sutures. Transparent corneal graft acceptance does not guarantee high visual acuity due to post-keratoplasty astigmatism. Surgical correction of astigmatism is performed in the long term post-operatively and isn't effective enough. We proposed this new surgical technique of keratoplasty with simultaneous implantation of ICCR and ICRS into the graft as close as possible to the visual axis of the eye, which can help make the postoperative astigmatism minimal both immediately after surgery and in the long term. The study proposes a new approach to intraoperative prevention of post-keratoplasty astigmatism. The technique is simple, safe and effective. Analysis of long-term outcomes is required before recommending this method for widespread use in clinical practice.

[A new surgical approach to pre-Descemet's endothelial keratoplasty]. / Novyi khirurgicheskii podkhod k predestsemetovoi endotelial'noi keratoplastike (PDEK).

PURPOSE: The study aimed to develop new surgical methods of obtaining and preserving pre-Descemet's endothelial keratoplasty (PDEK) graft in order to reduce the loss of donor material. MATERIAL AND METHODS: The study was conducted on 30 sclerocorneal discs, which were divided into three groups. The first group consisted of 10 sclerocorneal discs to which the standard PDEK graft preparation technique was applied. The second group consisted of 10 sclerocorneal discs, where the PDEK graft preparation was done using the optimized method 1 involving the KD Ring fixator, KD Base for PDEK, KD-30G needle with 5 mL syringe connected to the spring-load plunger, a preservative solution. The third group consisted of 10 sclerocorneal discs with PDEK graft prepared using the optimized method 2, which differed from the optimized method 1 in additional use of the KD artificial chamber for PDEK. When successfully acquiring the type-1 "big bubble", the dissected PDEK graft was preserved for further use in clinical practice using our own technique. RESULTS: In the first group, formation of the type-1 «big bubble¼ was achieved only in 5 out of 10 donor corneas (50%). Bubble ruptures happened in 4 cases (40%) and type-2 «big bubble¼ occurred in 1 case (10%). In the second group (optimized method 1) the type-1 «big bubble¼ was achieved in 9 cases (90%), while in the third group (optimized method 2) the type-1 «big bubble¼ was achieved in 10 cases (100%), proving the effectiveness of our PDEK graft preparation technique. CONCLUSION: We presented a novel surgical approach for acquiring and preserving PDEK graft that minimizes almost all the difficulties that surgeons face, including bubble rupture during pneumodissection and hydrodissection, formation of type-2 and mixed type bubble, and obtaining a PDEK graft of the required size; the details of the surgical technique have been refined. The proposed technique for graft preparation and preservation can be easily implemented in eye banks and is convenient for clinical practice.

Accessing Diverse Pyridine-Based Macrocyclic Peptides by a Two-Site Recognition Pathway.

Macrocyclic peptides are sought-after molecular scaffolds for drug discovery, and new methods to access diverse libraries are of increasing interest. Here, we report the enzymatic synthesis of pyridine-based macrocyclic peptides (pyritides) from linear precursor peptides. Pyritides are a recently described class of ribosomally synthesized and post-translationally modified peptides (RiPPs) and are related to the long-known thiopeptide natural products. RiPP precursors typically contain an N-terminal leader region that is physically engaged by the biosynthetic proteins that catalyze modification of the C-terminal core region of the precursor peptide. We demonstrate that pyritide-forming enzymes recognize both the leader region and a C-terminal tripeptide motif, with each contributing to site-selective substrate modification. Substitutions in the core region were well-tolerated and facilitated the generation of a wide range of pyritide analogues, with variations in macrocycle sequence and size. A combination of the pyritide biosynthetic pathway with azole-forming enzymes was utilized to generate a thiazole-containing pyritide (historically known as a thiopeptide) with no similarity in sequence and macrocycle size to the naturally encoded pyritides. The broad substrate scope of the pyritide biosynthetic enzymes serves as a future platform for macrocyclic peptide lead discovery and optimization.

Vexitoxins: conotoxin-like venom peptides from predatory gastropods of the genus Vexillum.

Venoms of predatory marine cone snails are intensely studied because of the biomedical applications of the neuropeptides that they contain, termed conotoxins. Meanwhile some gastropod lineages have independently acquired secretory glands strikingly similar to the venom gland of cone snails, suggesting that they possess similar venoms. Here we focus on the most diversified of these clades, the genus Vexillum. Based on the analysis of a multi-species proteo-transcriptomic dataset, we show that Vexillum species indeed produce complex venoms dominated by highly diversified short cysteine-rich peptides, vexitoxins. Vexitoxins possess the same precursor organization, display overlapping cysteine frameworks and share several common post-translational modifications with conotoxins. Some vexitoxins show sequence similarity to conotoxins and adopt similar domain conformations, including a pharmacologically relevant inhibitory cysteine knot motif. The Vexillum envenomation gland (gL) is a notably more recent evolutionary novelty than the conoidean venom gland. Thus, we hypothesize lower divergence between vexitoxin genes, and their ancestral 'somatic' counterparts compared to that in conotoxins, and we find support for this hypothesis in the evolution of the vexitoxin cluster V027. We use this example to discuss how future studies on vexitoxins can inform the origin of conotoxins, and how they may help to address outstanding questions in venom evolution.

[The Dizziness Handicap Inventory for quality control in the treatment of vestibular dysfunction]. / Dizziness Handicap Inventory in der Qualitätssicherung der Therapie vestibulärer Funktionsstörung.

Treatment of vestibular dysfunction remains a challenge for many otolaryngologists. This is true not only for establishing the differential neurotologic diagnosis, but also for treatment and treatment monitoring in vestibular dysfunction patients. Particularly quality control of therapeutic measures is generally poorly documented. The validated German version of the Dizziness Handicap Inventory (DHI) represents a viable option for evaluation and monitoring of treatment outcomes. In this study, patients who were treated at the Otorhinolaryngology Department of the University Hospital Aachen because of unilateral peripheral vestibular disorders were asked to complete the DHI before and after treatment. Posttherapeutic DHI scores were collected by telephone. Evaluation of the DHI scores underlined the significant benefit of treatment in 92%. Furthermore, the DHI prove to be useful for documentation of patients' disorders and treatment and thus for quality assurance.

Prevalence and genotype distribution of JC polyomavirus in urine from patients with hematological malignancies in Vietnam.

JC virus (JCV) causes progressive multifocal leukoencephalopathy in immunocompromised patients. The prevalence and genotype patterns of JCV vary between different geographical regions. This study was done to investigate the prevalence and genotype distribution of JCV in patients with hematological malignancies in Vietnam. A total of 48 urine samples were collected from patients with hematological malignancies. DNA was extracted and detection of JCV was by nested-polymerase chain reaction. Sequence analysis was obtained and a phylogenetic tree was constructed for genotyping of JCV. Twenty-seven (56.25%) urine samples tested positive for JCV. JCV genotype 7 was only observed in this study. Subtype analysis showed that JCV subtype 7A was the most commonly prevalent, followed by 7B1 and 7C1. Other subtypes were not detected in this population. There were no significant differences associated with age, gender, and biochemical parameters between patients with JCV and without JCV excretion in urine. The present study showed a high prevalence of JCV in the urine of patients with hematologic malignancies. The most common genotype found in this population was JCV subtype 7A.

Clinical determinants of early spontaneous conversion to sinus rhythm in patients with atrial fibrillation.

BACKGROUND: The current standard of care for acute atrial fibrillation (AF) focuses primarily on immediate restoration of sinus rhythm by cardioversion, although AF often terminates spontaneously. OBJECTIVE: To identify determinants of early spontaneous conversion (SCV) in patients presenting at the emergency department (ED) because of AF. METHODS: An observational study was performed of patients who visited the ED with documented AF between July 2014 and December 2016. The clinical characteristics and demographics of patients with and without SCV were compared. RESULTS: We enrolled 943 patients (age 69 ± 12 years, 47% female). SCV occurred within 3 h of presentation in 158 patients (16.8%). Logistic regression analysis showed that duration of AF <24 h [odds ratio (OR) 7.7, 95% confidence interval (CI) 3.5-17.2, p < 0.001], left atrial volume index <42 ml/m2 (OR 1.8, 95% CI 1.2-2.8, p = 0.010), symptoms of near-collapse at presentation (OR 2.4, 95% CI 1.2-5.1, p = 0.018), a lower body mass index (BMI) (OR 0.9, 95% CI 0.91-0.99, p = 0.028), a longer QTc time during AF (OR 1.01, 95% CI 1.0-1.02, p = 0.002) and first-detected AF (OR 2.5, 95% CI 1.6-3.9, p < 0.001) were independent determinants of early SCV. CONCLUSION: Early spontaneous conversion of acute AF occurs in almost one-sixth of admitted patients during a short initial observation in the ED. Spontaneous conversion is most likely to occur in patients with first-onset, short-duration AF episodes, lower BMI, and normal left atrial size.

Parametric attosecond pulse amplification far from the ionization threshold from high order harmonic generation in He.

Parametric amplification of attosecond coherent pulses around 100 eV at the single-atom level is demonstrated for the first time by using the 3D time-dependent Schrödinger equation in high-harmonic generation processes from excited states of He+. We present the attosecond dynamics of the amplification process far from the ionization threshold and resolve the physics behind it. The amplification of a particular central photon energy requires the seed XUV pulses to be perfectly synchronized in time with the driving laser field for stimulated recombination to the He+ ground state and is only produced in a few specific laser cycles in agreement with the experimental measurements. Our simulations show that the amplified photon energy region can be controlled by varying the peak intensity of the laser field. Our results pave the way to the realization of compact attosecond pulse intense XUV lasers with broad applications.

Parenting Teamwork: The Impact of a Fathering Intervention on Mothers and Infants in Vietnam.

A multifaceted, relationally focused intervention involving group and individual pre- and postnatal counseling, print resources, and community resources encouraged 390 fathers of newborn infants in Vietnam to responsively support mothers and work with them as a parenting team. Both partners completed questionnaires prebirth and 1-, 4-, and 9-months postbirth on measures of breastfeeding support, exclusive breastfeeding duration, relationship quality, and infant development. Compared to 412 comparison group couples, intervention couples evidenced greater father support, especially in terms of helping and responsiveness to the mother's needs. This support predicted longer exclusive breastfeeding duration, improved relationship quality, and higher levels of infant development at 9 months. Sensitively working together with mothers as a coordinated team enhanced couple's relationship functioning and improved children's developmental outcomes.

Unusual Polyhydroxylated Steroids from the Starfish Anthenoides laevigatus, Collected off the Coastal Waters of Vietnam.

Four new polyhydroxylated steroids 1-4 were isolated along with two previously known related steroids 5 and 6 from the methanolic extract of the starfish Anthenoides laevigatus collected off the coastal waters of Vietnam. Structures of new compounds were substantially elucidated by one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy and HRESIMS techniques. Heptaol 1 and hexaol 2 contain the common 5α-cholestane skeleton, while hexaol 3 and heptaol 4 have the rare among starfish steroid compounds 5ß-cholestane skeleton. Compounds 1, 5, and 6 do not show cytotoxic effects against normal JB6 Cl41 and cancer HT-29 and MDA-MB-231 cells, however they inhibit cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.

Uncovering sperm metabolome to discover biomarkers for bull fertility.

BACKGROUND: Subfertility decreases the efficiency of the cattle industry because artificial insemination employs spermatozoa from a single bull to inseminate thousands of cows. Variation in bull fertility has been demonstrated even among those animals exhibiting normal sperm numbers, motility, and morphology. Despite advances in research, molecular and cellular mechanisms underlying the causes of low fertility in some bulls have not been fully elucidated. In this study, we investigated the metabolic profile of bull spermatozoa using non-targeted metabolomics. Statistical analysis and bioinformatic tools were employed to evaluate the metabolic profiles high and low fertility groups. Metabolic pathways associated with the sperm metabolome were also reported. RESULTS: A total of 22 distinct metabolites were detected in spermatozoa from bulls with high fertility (HF) or low fertility (LF) phenotype. The major metabolite classes of bovine sperm were organic acids/derivatives and fatty acids/conjugates. We demonstrated that the abundance ratios of five sperm metabolites were statistically different between HF and LF groups including gamma-aminobutyric acid (GABA), carbamate, benzoic acid, lactic acid, and palmitic acid. Metabolites with different abundances in HF and LF bulls had also VIP scores of greater than 1.5 and AUC- ROC curves of more than 80%. In addition, four metabolic pathways associated with differential metabolites namely alanine, aspartate and glutamate metabolism, ß-alanine metabolism, glycolysis or gluconeogenesis, and pyruvate metabolism were also explored. CONCLUSIONS: This is the first study aimed at ascertaining the metabolome of spermatozoa from bulls with different fertility phenotype using gas chromatography-mass spectrometry. We identified five metabolites in the two groups of sires and such molecules can be used, in the future, as key indicators of bull fertility.

Occurrence of Melibiose-Containing Glycosphingolipids in a Sample of a Sponge-Coral Association (Desmapsamma anchorata/Carijoa riisei).

In our research on biologically active compounds from Vietnamese marine invertebrates, rare melibiose-containing glycosphingolipids were found in a sample of a sponge-coral association (Desmapsamma anchorata/Carijoa riisei). Melibiosylceramides were analyzed as constituents of some multi-component RP-HPLC fractions, and the structures of 14 new (1b, 3b, 4a-4c, 6a-6c, 8b, 9a, 9b, 10b, 11a, 11b) and five known (2b, 5a-5c, 7b) natural compounds were elucidated using NMR, mass spectrometry, optical rotation, and chemical transformations. These α-d-Galp-(1→6)-ß-d-Glcp-(1 ↔ 1)-ceramides (presumably sponge-derived compounds) were shown to contain phytosphingosine-type n-t17:0 (1), (6E)-n-t17:1 (2), i-t17:0 (3), n-t18:0 (4), (6E)-n-t18:1 (5), i-t18:0 (6), (6E)-i-t18:1 (7), i-t19:0 (8), (6E)-i-t19:1 (9), ai-t19:0 (10), and (6E)-ai-t19:1 (11) backbones N-acylated with saturated straight-chain (2R)-2-hydroxy C21 (a), C22 (b), and C23 (c) acids. Characteristic trends in the fragmentations of the terminal parts of tetraacetylated normal-chain and iso- and anteiso-branched sphingoid bases were observed using GC/MS. The total sum of melibiosylceramides and compound 5b caused a reduction in colony formation of human melanoma cells.

Six New Polyhydroxysteroidal Glycosides, Anthenosides S1 - S6, from the Starfish Anthenea sibogae.

Six new polyhydroxysteroidal glycosides, anthenosides S1  - S6 (1 - 6), along with a mixture of two previously known related glycosides, 7 and 8, were isolated from the methanolic extract of the starfish Anthenea sibogae. The structures of 1 - 6 were established by NMR and HR-ESI-MS techniques as well as by chemical transformations. All new compounds have a 5α-cholest-8(14)-ene-3α,6ß,7ß,16α-tetrahydroxysteroidal nucleus and differ from majority of starfish glycosides in positions of carbohydrate moieties at C(7) and C(16) (1 - 4, 6) or only at C(16) (5). The 4-O-methyl-ß-d-glucopyranose residue (2) and Δ24 -cholestane side chain (3) have not been found earlier in the starfish steroidal glycosides. The mixture of 7 and 8 slightly inhibited the proliferation of human breast cancer T-47D cells and decreased the colony size in the colony formation assay.

Enhanced sensitivity and responses to viologens from a whole-cell bacterial bioreporter treated with branched polyethyleneimines.

AIMS: Evaluate the use of polyethyleneimines (PEIs) as membrane permeabilizers to improve the responses and sensitivity of a bacterial bioreporter strain to viologens. METHODS AND RESULTS: The responses from E. coli str. EBS, i.e., E. coli BW25113 carrying plasmid pSDS, when exposed to five different viologens were characterized, as were the toxicities of seven different PEIS, including two linear and five branched species. Based on these results, benzyl viologen led to the greatest responses, and 0·8-kDa branched PEI (BPEI) was the least toxic of the PEIs tested and, therefore, both were selected for the subsequent tests. The bioluminescence and relative responses from E. coli str. EBS exposed to various concentrations of 0·8 kDa BPEI identified 400 mg l-1 as the optimal concentration. Using this concentration, tests were performed with all five of the viologens. CONCLUSIONS: The responses from E. coli str. EBS to the viologens were improved, with the maximum relative bioluminescence values increasing between 5·6 and 16·5-fold. The minimum detectable levels for four of the viologens were likewise improved 2- to 4-fold. SIGNIFICANCE AND IMPACT OF STUDY: Improving bacterial membrane permeability in a controlled manner using BPEIs can improve biosensing of toxic compounds, as well as be used in biofuel and bioenergy applications where membrane permeability to a solute is important.

Presence and extent of cardiac computed tomography angiography defined coronary artery disease in patients presenting with syncope.

BACKGROUND: In syncope patients, presence of coronary artery disease (CAD) is associated with poor prognosis. However, data concerning CAD prevalence in syncope patients without known cardiovascular disease are lacking. Therefore, the aim of this study was to investigate presence and extent of CAD in syncope patients. METHODS: We included 142 consecutive patients presenting with syncope at the outpatient cardiology clinic who underwent coronary computed tomography (CT) angiography. Syncope type was ascertained by two reviewers, blinded for coronary CT angiography results. Of the patients, 49 had cardiac syncope (arrhythmia or structural cardiopulmonary disease) and 93 had non-cardiac syncope (reflex [neurally-mediated], orthostatic or of unknown cause). Cardiac syncope patients were compared with matched stable chest pain patients regarding age, gender, smoking status, diabetes mellitus type 2 and systolic blood pressure. RESULTS: Distribution of CAD presence and extent in cardiac and non-cardiac syncope patients was as follows: 72% versus 48% any CAD; 31% versus 26% mild, 8% versus 14% moderate and 33% versus 7% severe CAD. Compared with non-cardiac syncope, patients with cardiac syncope had a significantly higher CAD presence and extent (p = 0.001). Coronary calcium score, segment involvement and stenosis score were also higher in cardiac syncope patients (p-values ≤0.004). Compared to the chest pain control group, patients with cardiac syncope showed a higher, however, non-significant, prevalence of any CAD (72% versus 63%) and severe CAD (33% versus 19%). CONCLUSION: Patients with cardiac syncope show a high presence and extent of CAD in contrast to non-cardiac syncope patients. These results suggest that CAD may play an important role in the occurrence of cardiac syncope.

MARCO variants are associated with phagocytosis, pulmonary tuberculosis susceptibility and Beijing lineage.

Macrophage receptor with collagenous structure (MARCO) has an important role in the phagocytosis of Mycobacterium tuberculosis (M. tuberculosis). We hypothesized that MARCO polymorphisms are associated with phagocytosis, tuberculosis (TB) disease susceptibility and presentation, and infecting lineage. We used a human cellular model to examine how MARCO genotype mediates the immune response; a case-control study to investigate tuberculosis host genetic susceptibility; and a host-pathogen genetic analysis to study host-pathogen interactions. Two MARCO heterozygous (AG) genotypes (single-nucleotide polymorphisms rs2278589 and rs6751745) were associated with impaired phagocytosis of M. tuberculosis trehalose 6,6'-dimycolate-cord factor and ß-glucan-coated beads in macrophages. The heterozygous genotypes of rs2278589 and rs6751745 were also associated with increased risk of pulmonary TB (PTB; rs2278589, P=0.001, odds ratio (OR)=1.6; rs6751745, P=0.009, OR=1.4), and with severe chest X-ray abnormalities (P=0.007, OR=1.6). These two genotypes were also associated with the Beijing lineage (rs2278589, P=0.001, OR=1.7; rs6751745, P=0.01, OR=1.5). Together, these results suggest that MARCO polymorphisms may regulate phagocytosis of M. tuberculosis and susceptibility and severity of PTB. They also suggest MARCO genotype and Beijing strains may interact to increase the risk of PTB.

The role of IL-1ß in Pseudomonas aeruginosa in lung infection.

This mini-review examines the role of the pro-inflammatory cytokine interleukin (IL)-1ß in the interaction of Pseudomonas aeruginosa and the host immune system during lung infection. Different studies show that the reduction of the inflammatory response, especially a decrease in IL-1ß, leads to a better outcome in acute lung infection with this bacterium. This includes a higher survival rate, reduced damage to the lung tissue and, in particular, a better clearance of the airways and the tissue of the lungs from P. aeruginosa.

Truncated Cables1 causes agenesis of the corpus callosum in mice.

Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are known to be involved in corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear. Here, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis to determine the genomic insertion site. Transgenes were inserted into chromosome 18 ∼12.0 Mb from the centromere. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1(TAS). Interestingly, Cables1(TAS) mRNA consisted of exons 1-3 of Cables1 and part of the transgene that encoded a novel truncated Cables1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1(TAS) in the fetal cerebrum, but not that of wild-type Cables1. To investigate whether a dominant negative effect of Cables1(TAS) or complete loss of function of Cables1 gives rise to ACC, we produced Cables1-null mutant mice. ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of Cables1(TAS) impairs callosal formation. Moreover, ACC frequency in Cables1(+/TAS) mice was significantly lower than that in Cables1(-/TAS) mice, indicating that wild-type Cables1 interfered with the dominant negative effect of Cables1(TAS). This study indicated that truncated Cables1 causes ACC and wild-type Cables1 contributes to callosal formation.

Development of solar concentrators for high-power solar-pumped lasers.

We have developed unique solar concentrators for solar-pumped solid-state lasers to improve both efficiency and laser output power. Natural sunlight is collected by a primary concentrator which is a 2 m×2 m Fresnel lens, and confined by a cone-shaped hybrid concentrator. Such solar power is coupled to a laser rod by a cylinder with coolant surrounding it that is called a liquid light-guide lens (LLGL). Performance of the cylindrical LLGL has been characterized analytically and experimentally. Since a 14 mm diameter LLGL generates efficient and uniform pumping along a Nd:YAG rod that is 6 mm in diameter and 100 mm in length, 120 W cw laser output is achieved with beam quality factor M2 of 137 and overall slope efficiency of 4.3%. The collection efficiency is 30.0 W/m2, which is 1.5 times larger than the previous record. The overall conversion efficiency is more than 3.2%, which can be comparable to a commercial lamp-pumped solid-state laser. The concept of the light-guide lens can be applied for concentrator photovoltaics or other solar energy optics.

Optimized Kalinnikov-Dinh technology for effective pre-Descemet's endothelial keratoplasty (PDEK) graft preparation and preservation.

We report an optimized Kalinnikov-Dinh technology for pre-Descemet's endothelial keratoplasty (PDEK) that involves the use of a ring fixator, base, 30G needle connected to a 5-ml syringe with a spring-loaded plunger, and storage media. Our method allows to minimize graft preparation failure and preserves the PDEK graft efficiently, by reducing complications associated with the formation of type 1 big bubbles, including bubble rupture, perforation of Descemet's membrane and endothelium, and formation of type 2 or mixed type of big bubbles, and may contribute to increasing the number of surgeons performing PDEK around the globe.