Cardiovascular Journal of South Africa (CVJSA) has made unprecedented strides in the past 18 months, based on steady progress since its inception in 1989.The Cardiovascular Journal of South Africa (CVJA), incorporating the CVJSA, in issue 5 of volume 18. It is now widely visible, not only in Africa, but also in North America, Europe and the Middle East.In October 2007 the Journal became the Currently, both CVJSA and CVJA are recorded in databases by their printed and on-line ISSN numbers for indexing and listing purposes. These data can be retrieved and viewed, providing an integrated statistical history, some of which will be reflected for the month of September 2008 on page 308 in this issue.Science Citation Index Expanded (SciSearch) and Journal Citation Reprints/Science Edition. This brings the CVJA to the forefront of international medical literature and will enable the development of an impact factor. Although the impact factor for assessing quality of information leaves much to be desired, it is still the benchmark used by institutions and funders as a guide to decision making on career advancement and allocation of funds.Most importantly, the CVJA has now been accredited, indexed and abstracted in We make a special appeal to all authors and readers of this Journal to support the publication to their utmost, in order to increase the citation rate. CVJA data are also visible in the Elsevier competitor citation resource, known as Scopus.The final issue of CVJA this year also marks the end of 20 years of production. Having come this far and achieved the above milestones, we have decided to celebrate the event early in 2009 with a celebratory historical issue, which has already been structured and is in the process of being compiled.The sustained interest, contributions and support of the many readers, authors and reviewers in the field of cardiovascular medicine in South Africa and other countries have made this remarkable achievement possible. This is the only quality journal of its kind on the African continent. Thank you to all.The celebratory events will also include a gala evening, where all are welcome. Please contact the Journal offices on cvjsa@cvjsa.co.za or Elsabe on (021) 976-8129 (mornings only) for an invitation. In particular, founding sponsors, editorial board members and reviewers will hopefully attend and the intention is to have at least one prominent editor from an overseas journal present.South African Journal for Diabetes and Vascular Disease will move to a new website.The front cover of CVJSA is being redesigned for added impact and a more modern appearance. The present website is also to be redesigned for the CVJA to make it more user friendly and functional. The sister publication, the As we face the future, we are aware of the economic stresses worldwide and must consider strategic ways to overcome this period of recession, which is expected to be of considerable duration.Pocket Guide to the Treatment of Heart Failure in a Multicultural Society (electronic and printed versions), which will be updated on an ongoing basis. We will consider the production of other guides on cardiovascular medicine in the future.Smaller projects of significance that we have undertaken to increase income include attendance at international congresses for the advance electronic reporting of international trial results, and the publication of the All in all, we have had a challenging but productive 20 years and thank our many supporters who have encouraged us to reach the highest standard possible.Season’s greetings. We wish you and yours a happy and prosperous new year.


In circulating influenza viruses, gradually accumulated mutations on the glycoprotein hemagglutinin (HA), which interacts with infectivity-neutralizing antibodies, lead to the escape of immune system . The antibody recognition is highly correlated to the conformation change on the antigenic sites (epitopes), which locate on HA surface. To quantify a changed epitope for escaping from neutralizing antibodies is the basis for the antigenic drift and vaccine development.We have developed an epitope-based method to identify the antigenic drift of influenza A utilizing the conformation changes on epitopes. A changed epitope, an antigenic site on HA with an accumulated conformation change to escape from neutralizing antibody, can be considered as a "key feature" for representing the antigenic drift. According to hemagglutination inhibition (HI) assays and HA/antibody complex structures, we statistically measured the conformation change of an epitope by considering the number of critical position mutations with high genetic diversity and antigenic scores. Experimental results show that two critical position mutations can induce the conformation change of an epitope to escape from the antibody recognition. Among five epitopes of HA, epitopes A and B, which are near to the receptor binding site, play a key role for neutralizing antibodies. In addition, two changed epitopes often drive the antigenic drift and can explain the selections of 24 WHO vaccine strains.Our method is able to quantify the changed epitopes on HA for predicting the antigenic variants and providing biological insights to the vaccine updates. We believe that our method is robust and useful for studying influenza virus evolution and vaccine development. Influenza viruses occur all over the world and cause significant morbidity and mortality . The surMost of methods measuring the antigenic variances on HA focused on amino acid position mutations, such as hamming distance  or phyloHere, we have proposed a method to identify the antigenic drift of influenza A by quantifying the conformation change of an epitope. Our method is able to predict antigenic variants of a given pair HA sequences which are often a vaccine strain and a circulating strain. Our model was evaluated to measure the antigenic drifts and vaccine updates on 3,331 circulating strains (from year 1982 to 2009) and to predict the antigenic variants on two data sets . These results demonstrate that our model is able to reflect the biological meanings and can explain the selections of WHO vaccine strains.Figure et al.[et al.[The changed epitope is the core of our method. Here, we defined a changed epitope as follows: an antigenic site (epitope) on HA with accumulated amino acid mutations induces the conformation change to escape from the neutralizing antibody. The conformation change of a mutation depends on its position on HA structure and the mutation rate during 40 years. A changed epitope can be considered as a "key feature" for measuring antigenic variants of a pair HA sequences. Here, a changed epitope can be used to predict antigenic variants and antigenic drifts for the selections of vaccine strains. The definition of five epitopes including 131 positions was proposed by Wilson et al. and Bushl.[et al..http://gemdock.life.nctu.edu.tw/influenza/File1.xls) with 125 HA sequences from Weekly Epidemiological Record (WER) [et al.[To describe and evaluate the ability of the changed epitopes for predicting antigenic variants, we collected hemagglutination inhibition (HI) assays, describing the antigenic variants and similar viruses of the current global influenza surveillance system. The HI assay describes whether one (e.g. circulating) strain will be recognized by an antibody against the vaccine strain. We collected 343 H3N2 virus HI assays (Data available at rd (WER) , World Hrd (WER)  and relard (WER)  (Table Srd (WER) . Among 3http://gemdock.life.nctu.edu.tw/influenza/File2.xls) from influenza virus resource [To study the antigenic drifts and WHO vaccine updates, we collected 3,331 HA sequences  as "antigenic variants". Epitopes A and B, which are near to the receptor binding site, often play the key role for escaping from neutralizing antibody. Here, the epitopes A and B (denoted as "B+") were regarded as "changed" if there are more than 2 and 3 mutations, respectively. For the pair A/Mississippi/1/85 and A/Leningrad/360/86  to measure the vaccine efficiency on year In this study, we followed our previous work to select the critical positions  having hCurrently, several methods measured a changed epitope to escape from neutralizing antibody . Here, wFigures For Model two, the average number of changed epitopes was 2.2 for these 34 similar viruses.  According to the distribution Figure , Model tModel three considered a changed epitope when the number of mutations on the 64 selected critical positions is more than 2. In Model two, the numbers of "antigenic variants" and "similar viruses" with ≥ 3 changed epitopes were 119 and 16, respectively Fig. . The aveThree HA/antibody complex structures can be used to provide structural evidences for the changed epitopes  Fig. S11. Among Among 72 "antigenic variants" pairs with one changed epitope based on Model three, 70 pairs change on epitopes A or B. The single changed epitope on A or B, which can cause "antigenic variants", agreed to HA/antibody complex structures and the experiments. The receptor binding site, surrounded by epitopes A and B, is a basis for HA protein for the neutralizing mechanism ,22 Fig..Based on this observation, the epitopes A and B play a key role for neutralizing antibodies. Model four based on Model three considered a pair HA sequences as "antigenic variants" when ≥ 2 changed epitopes or ≥ 1 changed epitope on A or B. In Model four, a pair HA sequences with ≥ 3 mutations on 64 critical positions for the epitope B is regarded as "antigenic variants". Thus, we annotated a virus-pairs with single changed epitope on A or B as "1+" type Fig. . For exaIn the HA/antibody structure complex (PDB code 1KEN ), the anWe utilized the changed epitopes to study the antigenic drift on 3,331 circulating strains ranging from 1982 to 2009 (38 influenza seasons). One of WHO surveillance network's purposes is to detect the emergence and spread of antigenic variants that may signal a need to update the composition of influenza vaccine  , our model detected 12 seasons with emerging antigenic variants (VR ≥ 0.5) and 10 of them followed by the update of WER strain in the next season Fig. . For exaTo observe the epitope evolution, Figure According to the distribution of antigenic variants of Model four Fig. , it is iAmong 225 "antigenic variants" pairs, 13 pairs have no changed epitopes (Table S3 in additional file This study demonstrates our model is robust and feasible for quantifying the changed epitopes. According to the distribution of antigenic variants in HI assays and HA/antibody complex structures, we found that two critical position mutations with high genetic diversity and antigenic scores can induce the conformation change of an epitope. Epitopes A and B, closing the receptor binding site of HA, play a key role for neutralizing antibodies. In addition, two changed epitopes often drive the antigenic drift and can be used to explain the selections of 24 WHO vaccine strains. We believe that our method is useful for the vaccine development and studying the evolution of human influenza A virus.The authors declare that they have no competing interests.Conceived and designed the experiments: JWH and JMY. Performed the experiments and analyzed the data: JWH and JMY. Wrote the paper: JWH and JMYThe supplementary informationClick here for file


The blue-light photo-reduction (conversion of oxidized flavin quinone via flavin semiquinone to fully reduced flavin hydroquinone) and dark re-oxidation of the flavins riboflavin and lumiflavin in starch (α-amylose) films was studied by absorption and luminescence spectroscopy. Blue-light photo-excitation caused an absorption, fluorescence, and phosphorescence decrease which recovered in the dark. The photo-reduction dark-oxidation cycle could be repeated. The efficiency of photo-reduction decreased with exposed excitation energy, and the speed of re-oxidation in the dark slowed down with time after excitation. The absorption did not fully recover. The fluorescence efficiency after a long time of storage in the dark increased beyond the initial flavin quinone fluorescence efficiency. Flavin photo-excitation is thought to cause starch-flavin restructuring (static fluorescence quenching center formation), enabling enhanced photo-induced starch to flavin electron transfer with subsequent flavin reduction and starch oxidation. In the dark, after light switch-off, thermal reversion of flavin reduction and starch oxidation occurred. In the calculations σa,Fl(λ = 450 nm) ≈ 4.7 × 10−17 cm2  / 2 is the mean transmission at the excitation wavelength λexp in the excitation energy density interval of wexp,1 to wexp,2. Using adjacent data points of wexp, χFl, and A, the φred (wexp) curves were determined for RF and LF which are displayed in the inset of the lower part of φred  = 0.2 ± 0.03 and φred  = 0.12 ± 0.03. At the end of exposure the quantum yield of photo-reduction lowered to φred ≈ 10−4 for both samples .The absorbance recovery of flavin in starch after light exposure is manifested in The absorption recovery and the flavin re-oxidation fit reasonably well to bi-exponential functions according toa = A / Aini (λ) is the absorbance ratio at wavelength λ and time trec = 0 after end of exposure. χFl(0) is the mole-fraction of retained oxidized flavin at end of exposure. τrec,f is the time constant of fast absorption recovery, and τrec,s is the time constant of slow absorption recovery. The coefficients of fast recovery, κa,f and κχ,f, and the coefficients of slow recovery, κa,s and κχ,s, are related by κχ,per = 1 − κχ,f − κχ,s. The obtained parameters for RF in starch are χRF(0) = 0.135, κχ,f(RF) = 0.077, τrec,f(RF) = 3.3 h, κχ,s(RF) = 0.50, τrec,s(RF) = 229 h, and κχ,per(RF) = 0.423. The obtained parameters for LF in starch are χLF(0) = 0.096, κχ,f(LF)= 0.11, τrec,f(LF) = 2.85 h, κχ,s(LF) = 0.72, τrec,s(LF) = 325 h, and κχ,per(LF) = 0.17.as is seen by the solid and dashed curves in rh ≈ 0.66 (for preceding few days the ambient relative humidity varied between 50% and 69%). The last filled triangle and the last dot were measured after storing the samples for four days in a desiccator (ϕrh < 0.1) and relative humidity during measurement of ϕrh ≈ 0.45.The absorbance recovery of RF in starch after the second cycle of light exposure is shown by the dotted-line connected dots in the top part of exp = 455 nm, excitation wavelength for fluorescence detection λF,exc = 450 nm). The left figures belong to RF in starch and the right figures belong to LF in starch. The top parts show the light exposure situation, and the bottom parts show the situation of fluorescence recovery in the dark. The depicted fluorescence spectra are normalized to the initial fluorescence signal height at the wavelength of maximum fluorescence emission, i.e., SF(λ)/SF,ini is displayed  = 523 nm, λF,max(LF)= 525 nm). The shape of the fluorescence spectra is approximately independent of exposed light energy density and time of recovery. The fluorescence signal height decreased with light exposure starting already at very low light exposure. The fluorescence emission recovered after light exposure even beyond the original fluorescence signal height (see below).The evolution of spectrally corrected fluorescence spectra of previously unexposed RF and LF in starch due to light exposure and dark recovery is shown in F = ∫ SF (λ)dλ, with exposed energy density wexp. The curves are normalized to the total initial fluorescence signal, ∑ F,ini = ∫ SF,ini (λ)dλ, (unexposed samples). Comparing the total fluorescence signal decrease with the absorbance decrease shown in wexp < 10−3 J·cm−2) where the absorbance decrease was still negligible. For higher exposed energy densities (wexp > 10−3 J·cm−2) the total fluorescence signal decreased in accordance with the reduction of flavin quinone to flavin hydroquinone  is plotted. The curves were obtained by normalizing the total fluorescence signals of the top part of φF,ini(RF/starch) = 0.37 and φF,ini (LF/starch) = 0.36. The decrease of fluorescence quantum yield with light exposure indicates increased fluorescence quenching of flavin quinone in the course of exposure (photo-induced quenching center formation by starch—flavin restructuring). At the end of exposure the total fluorescence signal was stronger decreased than the absorbance or the fraction of retaining flavin quinone because the flavin quinone specific fluorescence quantum yield decreased with wexp.In the lower part of φF,Fl,  of ribofφqc, of flavin-starch quenching center formation at low exposed energy density (wexp < 10−2 J·cm−2) may be estimated from the decrease of fluorescence efficiency with absorbed excitation light. It isThe quantum efficiency, qc is the length-integrated number density increment of formed quenching centers, and Δnph,abs is the corresponding number density of absorbed photons. At wexp, ΔN qc may be expressed by the amount of Fl component specific fluorescence quantum yield decrease ΔφF,Fl according towhere ΔN wexp isThe corresponding number density of absorbed photons at φqcversus wexp for RF and LF in starch are shown in the inset of the upper part of φqc ≈ 10 at low exposed energy density (wexp ≤ 10−5 J cm−2) and decreases to φqc ≈ 0.01 at wexp ≈ 0.01 J cm−2. This result shows that initially one flavin molecule excitation caused a starch—flavin structure optimization (starch restructuring) which changed about 10 normal fluorescing flavin molecules, Fln, in inert starch environment to non-fluorescent flavin molecules, Flqc, in fluorescence quenching active starch environment (flavin–glucose quenching center formation). The total number density of flavin quinones is NFl = NFln + NqcFl. The quantum efficiency of flavin-starch quenching center formation decreased with increasing exposed energy density showing that the starch polymer rearrangement was getting more and more difficult. For wexp > 10−2 J·cm−2Nqc(wexp)/NFl(wexp) became rather constant and even decreased over a certain energy density range for RF in starch where φF,Fl increased with wexp. The oxidized flavin specific fluorescence quantum yield φF,Flversus sample absorbance A(450 nm) is shown in the inset of the bottom part of The calculated curves of The dotted line connected dots in the top part of F/∑F,ini, is depicted. The fluorescence recovery fits reasonably well to bi-exponential functions  with the same recovery times, τrec,f and τrec,s, as found for the absorbance recovery  before measuring (relative humidity in laboratory during measurement was ϕrh ≈ 0.45).The fluorescence recovery of RF in starch after a second cycle of light exposure is shown by the dotted-line connected dots in the top part of φF,Fl, of flavin quinone on the dark time after first light exposure is depicted in the lower part of φF,Fl(0) ≈ 0.21, and it increased to φF,Fl(∞) ≈ 0.60. The initial fluorescence quantum yields were φF,RF,ini = 0.37 and φF,LF,ini = 0.36  with χn = 0.6, τF,n = 4.57 ns, χqc = 1 − χn = 0.4, and τF,qc = 1.5 ± 0.5 ps.Additionally, the time-resolved fluorescence of LF in starch film was measured before light exposure and after dark recovery with the mode-locked laser system and an ultrafast streak-camera  behavior was investigated. Delayed luminescence spectra and signal decay traces were measured. RF in starch and LF in starch behaved similar. Below in exp = 455 nm with different exposed energy densities, and the bottom part shows spectra belonging to different times of recovery in the dark. The spectra are normalized to the initial luminescence signal at 650 nm, i.e., SDL(λ)/SDL,ini(650 nm) is presented. The emission band peaking around 650 nm belongs to phosphorescence, and the emission band peaking around 530 nm belongs to delayed fluorescence (for details see [trec = 2655 h was measured at ϕrh = 0.66 after a few days of high humidity in the range of 50% to 69% in the laboratory. The strong reduction of delayed luminescence at high relative humidity is clearly seen. Above ϕrh = 50% the starch films begin to become slightly permeable to oxygen [3Fln* + 3O2 →1 Fln + 1O2*. The curve belonging to trec = 2740 h was measured at ϕrh = 0.45 after storing the sample for four days in a desiccator (ϕrh < 0.1). The delayed luminescence spectrum recovered nearly to the initial spectrum. The delayed fluorescence peak became similar high as the phosphorescence peak (some enhancement of delayed fluorescence).In ails see ). The spo oxygen  and the o oxygen  with sofo oxygen ,50 3Fln*pr = 530 nm  and at λpr = 645 nm  versus exposed energy density at λexp = 455 nm and versus recovery time in the dark is shown in A(450 nm)/Aini(450 nm) on exposed energy density and on time after exposure is included (dotted line connected diamonds). The delayed luminescence signal followed the exposure dependence and the recovery dependence of the fluorescence signal. For the second last data points in the bottom part of φDL,Fl, reached higher values than the initial unexposed sample. This behavior is the same as was found for the fluorescence. Both the fluorescence and the low-humidity delayed luminescence efficiency is proportional to the mole fraction of normal fluorescing flavin quinone χn.The development of the delayed luminescence at λn of normal fluorescing flavin quinone and the dissolved molecular oxygen dependent delayed luminescence lifetime shortening. The curve measured at trec = 2655 h under high relative humidity conditions shows a strong phosphorescence lifetime shortening. For the other curves the temporal decay curves have a similar shape. The curve measured at trec = 2740 h under low relative humidity conditions gave slightly higher luminescence signal than the initial unexposed sample.In n. The presence of flavins Flqc in quenching centers changes the radiative S1-state lifetime . The inclusion of the static fluorescence quenching causes the correction of a few parameters for RF in starch and LF in starch to: τrad,S1 (RF) = 7.2 ns, σ̄a, S0−S1 (RF) = 1.34 × 10−17 cm2, σa,max(RF) = 7.2 × 10−17 cm2, τF(LF) = 4.57 ns (new measurement because of some degradation of previous sample), τrad,S1 (LF) = 7.6 ns, σ̄a, S0−S1 (LF) = 1.28 × 10−17 cm2, σa,max(LF) = 6.85 × 10−17 cm2, φISC(LF) = 0.38, kISC(LF) = 8.32 × 107 s−1, and kT1,S1 (LF) = 1.13 s−1.The data analysis of unexposed RF and LF in starch films was carried out in  assumingn and flavins Flqc forming flavin-glucose fluorescence quenching centers. The proposed photocyclic dynamics and inter-conversion of Fln and Flqc is illustrated in The flavin behavior in starch may be described by a two-component system consisting of normal fluorescing flavins Flqc by photo-excitation of normal fluorescing flavin quinone Fln is illustrated in the top part of n leads to radiative relaxation, internal conversion, intersystem-crossing, back-intersystem-crossing (delayed fluorescence), radiative triplet-singlet relaxation (phosphorescence), and non-radiative triplet relaxation to the singlet ground-state. The energy released by non-radiative decay and excited-state charge distribution differences compared to the ground-state are thought to cause starch re-conformation to flavin-glucose fluorescence quenching center formation (Flqc) on the time range of phosphorescence decay . It should be noted that in the prepared flavin doped starch samples there were already fluorescence quenching centers present before light exposure.The formation of flavin-starch fluorescence quenching centers Flqc is thought to cause a reductive electron transfer [2 to flavin Fl according toThe photo-excitation of Fltransfer ,51 from − and glucose radical cation G(OH)2·+ is formed. The electron transfer quenches the fluorescence . Fl·− dominantly relaxes back to Flqc by charge recombination. Part of Fl·− converts to neutral flavin semiquinone FlH· by proton transfer (quantum efficiency ≥ φred) according towhereby flavin quinone radical anion Fl·· to FlH·* and causes subsequent electron transfer to flavin semiquinone radical anion according toThe incident light excites FlHA second proton transfer from the single oxidized glucose radical cation to the flavin semiqinone radical anion leads to flavin hydroquinone and oxy-glucose (oxystarch) according to2 formation is φred. In the dark there occurs thermal flavin hydroquinone re-oxidation to flavin quinone by oxystarch reduction to normal starch and a starch restructuring.The total quantum efficiency of FlH2 is visualized in The flavin reduction from Fl to FlH2 to LF might be caused by oxygen [2 back re-oxidation in the dark is less complete than for LFH2 (no back reduction of the oxidized ribityl part is indicated). In the starch film forming process by solution heating [ϕrh < 50%) are air impermeable  are involved in the meta-stable FMN-C4a-cysteinyl adduct formation ,55. The ·− or FMN·− with protein-flavin hydrogen-bond reorganization during the sub-nanosecond Fl·− charge-recombination lifetime giving the signaling state (light-adapted state) for effector domain activation [· and anionic flavin hydroquinone FlH− may take place with partial re-oxidation after light switch-off [In BLUF proteins ,16,19,56tivation ,57,59. Iitch-off ,57,59. Iitch-off ,56–59.− which is stabilized over a time range of seconds by positive charge separation along a Trp triade [·− may further reduce to FADH· and FADH− [In cryptochromes the primary receptor state—signaling state photocycle is given by photo-induced Tyr or Trp electron transfer to FAD forming FAD·p triade ,24,25,60nd FADH− . The rednd FADH− .The photo-induced formation of reversible dye radicals in the triplet state by application of reducing agents in aerobic liquid solutions was reported to be a frequently occurring phenomenon . It was rh < 0.5) are impermeable to oxygen and the retained oxygen in starch is immobile. Therefore flavin re-oxidation by oxygen in starch under dry ambient conditions is thought to be of minor importance.In the flavin doped solid starch films studied here, the starch host acts as reducing agent for photo-excited flavin. The thereby generated oxystarch acts as oxidizing agent for reduced flavin in its ground-state. In the case of riboflavin in starch hydrogen abstraction from the ribityl chain ,43,65–68MRF = 376.36 g·mol−1), lumiflavin (MLF = 256.3 g·mol−1), and starch  were purchased from Sigma-Aldrich and used as delivered. The solvent water was de-ionized in a Millipore water purifier and used in this form. The starch films were prepared from flavin-water and starch-water solutions on fused silica plates . The thickness of the starch films was approximately 36 μm. The flavin concentration in the starch films was roughly 8 × 10−3 mol·dm−3. Details of sample preparation are reported in [Riboflavin . The excitation light intensity was varied with optical neutral density filters (from Schott). The excitation intensity was measured with a power meter (Model PD 300-UV-SH photodiode detector head with NOVA power monitor from Ophir).The absorption spectra were measured with a spectrophotometer (Cary 50 from Varian). The fluorescence spectra, delayed luminescence spectra, and delayed luminescence lifetimes were measured with a fluorimeter (Cary Eclipse from Varian). Experimental details are found in .tres ≈ 500 ps. The fluorescence emission in front-face collection arrangement was directed to the detector via a broadband interference filter (transmission range 500 nm–600 nm) and a magic angle polarizer . For higher fluorescence signal time resolution (tres ≈ 10 ps) a streak camera  was used [Fluorescence lifetimes of the samples were measured using a mode-locked titanium-sapphire laser oscillator amplifier system (Hurricane from Spectra-Physics) for sample excitation . For fluorescence detection in most cases a micro-channel-plate photomultiplier (type R1564U-01 from Hamamatsu) was used in connection with a fast digital oscilloscope (LeCroy 9362) giving a time resolution of was used .Photo-excitation of the flavins riboflavin and lumiflavin in solid starch films caused repeatable reversible reduction . The flavin doped starch films may be considered as artificial flavin based blue-light photoreceptors since they exhibit a similar flavin reduction—re-oxidation photocycle dynamics as the natural biological blue-light photoreceptors . The role of the amino acid residues Cys, Tyr and Trp in the proteins of natural flavin-based blue-light photoreceptors is taken over in the artificial flavin doped starch films by the reducing action of the glucose repeat units of starch on photo-excited flavins and by the oxidizing action of di-aldehyde groups in the formed oxystarch on ground-state flavin in the reduced form.φqc, and the excitation energy density dependent quantum yield of flavin hydroquinone formation, φred. Similar photo-reduction and re-oxidation behavior is expected for FMN and FAD in starch.A quantitative analysis of the photo-reduction dynamics of riboflavin and lumiflavin in starch was carried out determining the excitation energy density dependent quantum efficiency of flavin-starch photo-reduction center formation,


A variety of complications related to endovascular procedures are being reported. We present an annoying complication implying complete migration of stent-graft of subclavian artery into the aortic lumen.A 61 years old man with abdominal endovascular aortic stent-graft inserted previously, had a saccular aneursym of descending aorta distal to left subclavian artery and a small dissection flap with saccular aneursym originating from left subclavian artery. Since the patient refused to have any surgical procedure for left subclavian artery revascularisation, thoracic aortic-stenting for descending aorta just below the subclavian origin and a second stent-graft for left subclavian artery just above the orifice was planned. Coverage of left subclavian artery was not preferred aiming to avoid possible cerebrovascular events. During intervention, stent-graft to be inserted inside subclavian artery migrated totally into aortic arch in a perpendicular fashion.Bilateral femoral and left brachial artery accesses were done. A 16x41mm stent-graft was inserted inside left subclavian artery. While insertion with balloon dilatation, stent-graft migrated distally with the balloon forming a partial prolapse into aortic arch. We tried several endovascular maneuvers including inserting a second graft into subclavian artery to stabilize the first one or trying to press this graft with the one inserted into descending aorta, but unfortunately the stent totally migrated into aorta. We catheterized migrated stent with balloon catheter and were able to move the graft with inflated balloon fixing it. Stent-graft and inflated balloon were pulled slowly back together to proximal part of left arm of the previously inserted abdominal aortic stent-graft and stabilized it carefully. Control DSA showed settled graft without any stenosis.We present a bothering complication and an endovascular solution we performed without complication.


P < .001), while SPAr had longer arterial ischemia 103 ± 42 min (P = .0004). One-year patient's and graft survival were, respectively, 89% and 95% versus 94% and 100% (P = .29). At median followup of 13 ± 6 versus 14 ± 7 months biliary complications were anastomotic stenosis in 15% versus 19% (P = .78), and intrahepatic nonanastomotic biliary strictures in 26% versus none (P = .01), respectively, in SPAr and CPAr. CPAr reduces the incidence of intrahepatic biliary strictures by decreasing the duration of arterial ischemia.We prospectively compared sequential portal-arterial revascularization  versus contemporaneous portal-hepatic artery revascularization  in 40 consecutive liver transplantation (LT). There were no differences in the demographics characteristics, MELD score, indication to LT, and donor's parameters between the two groups. CPAr had longer warm ischemia 66 ± 8 versus 37 ± 7 min ( Sequential portal-arterial revascularization (SPAr) of the graft is the most common technique in liver transplantation (LT), based on the experience that portal blood flow is sufficient for adequate liver function , 2. The P = .83). All grafts were whole liver except one extended right liver split including segment one in group 1. Portal vein and arterial anastomosis between the graft and the recipient have been always performed with terminal-terminal interrupted suture. In the SPAr group the graft was revascularized through the portal vein, and the hepatic artery was subsequently performed; the time interval between portal and arterial revascularization was recorded and defined as arterial ischemic time. In the CPAr the graft was revascularized simultaneously through the portal vein, and hepatic artery. Cold and warm ischemia time were determined, respectively, as the period between cross-clamp and graft inside the abdomen and between the graft in the abdomen and reperfusion. Reperfusion syndrome was defined as a drop in mean arterial pressure greater than 30% within 1 minute from reperfusion of the graft and lasting more than 5 minutes. Primary nonfunction (PNF) was defined as the need for retransplantation or death due to graft failure within 7 days in absence of technical problems. Delayed graft function (DGF) was defined as AST or ALT peak greater than 2000 U/ml within 72 hours from LT. Biliary complications were analyzed when clinically evident or through magnetic resonance cholangiopancreatography (MRCP) every 6 months after transplantation in asymptomatic patients. From June 2008 to December 2009, 40 consecutive LT from heart-beating donors were randomized 1 : 1 to SPAr  or CPAr . No differences between the groups for indications to OLT have been evidenced during statistical analysis. Cases requiring an aortohepatic bypass for arterial revascularization, and reOLT were excluded from the analysis. The LT was always carried out by piggy-back technique in all cases. The bile duct was reconstructed in all cases by termino-terminal duct to duct anastomosis; the T-tube was used, respectively, in 32% versus 29% of cases  years old and 74% versus 75% (P = .93) in group 1 and 2. No differences in the indication for LT were encountered with HCC as the main indication in 32% versus 42%, HCV in 26% versus 24%, and alcohol in 16% versus 19% (P = ns). The MELD score was 17 ± 7 versus 14 ± 5 (0.12). Donor's age was, respectively, 52 ± 18 versus 51 ± 15 years old in groups 1 and 2 (P = .85). Twenty-one percent of the grafts in groups 1 versus 19% in group 2 had macrovacuolar steatosis greater than 15% (P = .92). Duration of LT was not different between groups 1 and 2  as well as cold ischemia time , while warm ischemia time was statistically significant longer in the CPAr group . Arterial ischemic time in the SPAr group was statistically significant longer than warm ischemia time in the CPAr group . Units of blood and amount of plasma transfusion were, respectively, 6 ± 4 versus 7 ± 4 (P = .60) and 1,300 ± 1,175 ml versus 2,382 ± 1,853 ml (P = .045) in groups 1 and 2. There were no PNF in both groups; DGF was diagnosed in 10% versus 9% in groups 1 and 2 (P = .91). ICU and total hospital stay were, respectively, 7 ± 4 versus 5 ± 2 and 17 ± 5 versus 18 ± 6 days, respectively, in groups 1 and 2 (P = .12 and P = .68) (P = .29 and P = .53). At a median follow-up of 13 ± 6 versus 14 ± 7 months (P = .71) biliary complications diagnosed were anastomotic stenosis in 15% versus 19% (P = .78), and intrahepatic nonanastomotic biliary strictures in 26% versus none (P = .01), respectively, in SPAr and CPAr.Recipient's age and male gender were, respectively, 52 ± 9 versus 56 ± 9 (P = .68) . AST andP = .68) , total b5% P = .9 in groupIn all cases biliary stenosis occurred within 1 year after OLT, and these were successfully treated through ERCP when anastomotic  and through PTC (5 cases out of group 1) when intrahepatic nonanastomotic strictures have been diagnosed.The most commonly used procedure for revascularization of the liver graft is initial reperfusion via the portal vein and subsequent reconstruction of the hepatic artery –7. The d


Welcome to INIC 12 in the Republic of San Marino!I first would like to thank Paolo Pasini and his colleagues for their generous hospitality and work as hosts of INIC 12 here in the Republic of San Marino—it is sincerely and greatly appreciated.In most countries in the world the effective integration of health and social care has remained either an unfulfilled dream or an unrealistic ambition. This conference, that brings together leading-edge case examples of how this has been achieved, will undoubtedly show that local leadership, vision and commitment can overcome the many obstacles to integrated care. It is apt that the Republic of San Marino should host us since we will have much to learn from their approach and philosophy to care than overcomes traditional boundaries and places the patient and users health and well-being at its heart.International Foundation for Integrated Care (IFIC) was instituted. Its mission statement is as follows:Talking of ambition, in October 2011 the The International Foundation for Integrated Care is a network that crosses organisational and professional boundaries to bring people together to advance the science, knowledge and adoption of integrated care policy and practice. The Foundation seeks to achieve this through the development and exchange of ideas among academics, researchers, managers, clinicians, policy-makers and users and carers of services throughout the World.International Journal of Integrated Care (IJIC) as well as for the initiation and delivery of INIC’s Annual Congresses.Over the next year, this new Foundation—led by a Founding Board from across 12 countries—will develop a range of activities in support of its mission. This will include taking over responsibility from the Julius Centre, University Medical Centre (UMC) Utrecht for the funding of the scientific journal the I would like to take this opportunity to personally thank UMC Utrecht for its 12-year sponsorship and support of these activities, and also give a special word of thanks to Prof. Guus Schrijvers—without whom neither the journal or network would have been born—and his colleagues Clarine Sies and Inge Kuurman for their personal commitment as conference organisers.As the Foundation moves forward, it will also seek new opportunities including: a new website; a bi-annual World Congress (the first to be hosted in Singapore in 2013); the development of educational programmes; the creation of a knowledge-centre or observatory to support the Foundation as an expert international resource on integrated care; and to partner with other organisations in undertaking leading-edge research.This is an ambitious vision, but one which I hope you might share and wish to get involved with!I wish all of you an enjoyable and successful conference.


The multimedia launch was accompanied by Gustav Holst's Jupiter, the Bringer of Jollity. This is most fitting as applying advanced imaging and interventional techniques to improve the quality of health heralds joy to the world.As stated in the first issue, biij.We are especially interested in exploiting electronic publishing: online submission, online review, and online publication. Although numerous online journals have been published very few have taken advantage of the multimedia capabilities such as audio, video, animation, and simulation -3. We arbiij, namely Asian Breast Diseases Association (ABDA) Third Teaching Course: Advances in the Management of Breast Diseases, Kuantan, Malaysia (28-30 May 2005) and the 2nd International University of Malaya Research Imaging Symposium: Fundamentals of Molecular Imaging, Kuala Lumpur, Malaysia (16-17 July 2005).The abstracts of two international meetings have been archived in the first issue of the We are interested in archiving various abstracts and proceedings of scientific meetings. This serves as a depository of the various high-quality transactions.Since its inception there have been a total of 3,136 page loads or visits to the journal web site; made by 640 first-time visitors and 269 returning visitors (as of 1 October 2005). We are pleased to have an international readership including Argentina, Australia, Austria, Canada, Chile, China, Egypt, Germany, Hong Kong, India, Indonesia, Japan, Jordan, Korea, Philippines, Singapore, Spain, Thailand, United Arab Emirates, United Kingdom, United States, Uruguay, and Vietnam.Some of our readers have commented on its professional and polished appearance. From a modest start with four papers in the first issue we now have six papers in the second issue. Although this is an excellent start for a new journal we can do better.We have already planned for special focus issues on radiotherapy, informatics, intervention and PET/CT. Well known researchers and medical scientists have been invited to write the guest editorials. We invite you to contribute.biij is to attain the international standard and follow a stringent peer-review process. Our ultimate aim is to be indexed by various international bibliographic databases.The aspiration of biij has adopted the Uniform Requirements for Manuscripts Submitted to Biomedical Journals: Writing and Editing for Biomedical Publication  published by the International Committee of Medical Journal Editors [ Editors . PotentiThe success of a journal would have not been possible without the hard work of many people, including the Associate Editors, International Advisory Board and the Editorial Board, as well as all of the referees who take time to review the manuscripts. Finally we must not forget the corporations who sponsor the journal. Thanks to all of you.Each of you also has a vital role to play in the further development of the journal by publicizing it among your friends and colleagues, submitting manuscripts for consideration to be published and supporting the sponsors.We invite you to explore this innovative resource, and provide us with ideas and comments.


Increasingly smaller structures are gaining technological and economic importance. The scope of applications ranges from high-performance materials and coatings to car paints and catalysis, from bionic surfaces to medical applications, from nano-electronics to data storage devices, to mention only a few examples.Nanotechnology is considered one of the key technologies of the 21Rapid progress in nanotechnology is driven by two major factors. On the one hand, scientific progress allows the development of completely new products, processes and technologies. On the other hand, further progress in existing key technologies increasingly depends on the understanding and control of functional structures on the nanometer scale. Frequently, technological progress is hampered by a lack of understanding of structures on this length scale. To be successful, nanotechnology has to rely on a close cooperation of a number of different disciplines – including physics, chemistry and biology as well as engineering sciences and materials research.At the same time, the nanosciences are a vivid and rapidly developing area of research, continuously finding and understanding new phenomena and effects related to the small length scale. Things do not only become smaller, they behave differently, opening fascinating perspectives for new science and novel applications and technologies. Nano-photonics, quantum electronics, the lotus effect and the fascinating properties of graphene are only a few well-known examples.The Beilstein Journal of Nanotechnology provides a platform for the effective exchange and dissemination of cutting-edge results in the broad area of nanosciences and nanotechnology, including nanoscale physics, chemistry and biology, as well as engineering sciences, surface sciences, materials research and semiconductor technologies. It includes synthetic approaches and experimental work as well as computational nanoscience and theoretical aspects.The Beilstein Journal of Nanotechnology is unique in several respects:It is both an Open Access journal and a printed journal.It is freely accessible worldwide – thanks to the Open Access approach.There are no publication fees for the authors and no costs for the readers.The copyright of the articles and figures is retained by the authors – they are allowed to use articles or the material contained in their articles freely, e. g., for inclusion on their website.Who pays? – The Beilstein Journal of Nanotechnology is completely funded by the Beilstein-Institut (http://www.beilstein-institut.de), a prestigious non-profit foundation for the advancement of science. The Beilstein-Institut has a long tradition and history in scientific publishing, and a high reputation, especially in the area of chemical sciences, dating back to 1951.There is no commercial interest behind the Beilstein Journal of Nanotechnology or the Beilstein-Institut. The aim of the journal is to support scientists in the field of nanoscience and nanotechnology and to provide the widest possible platform for cutting-edge research.All articles are peer-reviewed. The main criteria for acceptance of an article for publication are high quality, originality, novelty and importance.The members of the Editorial Board and the Advisory Board are leading scientists in the field of nanoscience and nanotechnology worldwide, including Jean-Marie Lehn and Sir Harold Kroto as Nobel laureates.In this way, the Beilstein Journal of Nanotechnology, together with its sister journal, the Beilstein Journal of Organic Chemistry (http://www.bjoc.org), provides unique opportunities for the rapid and most effective dissemination of novel results. In addition to submitted articles, the concept of Thematic Series provides collections of invited, peer-reviewed contributions on selected focal areas of current interest, written by leading scientists in the specific field. If you are interested to initiate a Thematic Series, please contact the Editor-in-Chief.It is my honour to thank all those who have contributed and who will contribute to the success of this journal. I would like to thank the authors of the articles – and our referees, renowned researchers in the field of nanotechnology, who ensure the scientific quality of the journal. Many thanks go to the members of the Advisory Board and the Editorial Board for their excellent support. Above all, and also on behalf of the authors, the Advisory Board and the Editorial Board, I would like to thank the Beilstein-Institut for launching and funding this unique journal.It is my pleasure to invite you to become an active part in our journal and to profit from its advantages – as readers, as authors and as editors of Thematic Series.Thomas SchimmelEditor-in-ChiefKarlsruhe, November 2010


To evaluate the results of intravitreal expansile gas injection, with or without recombinant tissue plasminogen activator (rtPA), followed by intravitreal bevacizumab injection for treatment of submacular hemorrhage (SMH) secondary to neovascular age-related macular degeneration (AMD).In this interventional case series, 5 eyes of 5 patients with SMH secondary to choroidal neovascularization (CNV) due to neovascular AMD were treated with 0.3 cc intravitreal SF6 (and 50 μg of rtPA in two eyes), followed by face-down positioning; 24 hours later, 1.25 mg of bevacizumab was injected intravitreally. Main outcome measures included displacement of SMH and best corrected visual acuity (BCVA).Mean patient age was 75.6±9.2  years, mean duration of symptoms was 6.4±3.2  days, and mean number of bevacizumab injections was 1.8 . Mean preoperative BCVA was 1.28±0.27 logMAR which improved significantly to 0.57±0.33 logMAR at 12 months (P=0.042). SMH displacement occurred in all eyes, and visual acuity improved and remained stable during the follow-up period of 12 months.Intravitreal expansile gas injection, with or without rtPA, followed by intravitreal bevacizumab injection, seems to be an effective modality for SMH displacement and treatment of the underlying CNV in neovascular AMD. Thick submacular hemorrhage (SMH), particularly in patients with neovascular age-related macular degeneration (AMD), is generally associated with poor visual prognosis. Retrospective natural history studies have demonstrated that final visual acuity (VA) in AMD patients with thick blood beneath the fovea is rarely better than 20/200.Several reports have demonstrated the ability of an intravitreal gas bubble, withThis non-randomized interventional case series includes 5 eyes with SMH due to neovascular AMD that received intravitreal injection of expansile gas with or without rtPA followed by bevacizumab. Pre- and postoperative ophthalmologic examinations included determination of best corrected VA using a Snellen chart, slitlamp biomicroscopy, and indirect ophthalmoscopy. All patients were examined preoperatively and were followed for 12 months after the procedure. Color fundus photography, fluorescein angiography (FA), and optical coherence tomography (OCT) were performed. All SMHs were of sufficient thickness to induce significant elevation of the entire neurosensory retina, completely obscuring the underlying choroidal vascular pattern on biomicroscopic examination. Each patient gave informed consent to this novel treatment approach after the nature of the disease, its natural course and alternative treatment modalities had been explained.The procedure was performed under sterile conditions in the operating room. Intravitreal injection was performed 3.75 mm and 3 mm behind the limbus in phakic and pseudophakic eyes, respectively, using a #30 gauge needle. First, commercial rtPA, a lyophilized material, was diluted with balanced salt solution to a concentration of 1 mg ml in polypropylene syringes. Then, 0.05 ml of the preparation, equal to 50 μg rtPA, was injected intravitreally. Subsequently, 0.3 ml of pure sulfur hexafluoride (SF6) gas was injected into the vitreous cavity. All patients were instructed to maintain face-down position until the next day. Twenty-four hours later, intravitreal injection of 1.25 mg/0.05 ml bevacizumab was performed in a similar fashion and under the same setting as described above.Patients received reinjection(s) of bevacizumab when recurrent CNV was noticed. Recurrence was defined as a decrease in VA associated with new foci of subretinal hemorrhage or intraretinal fluid detected by OCT, and leakage noted by FA.Patient characteristics are shown in Case 1 is presented below as an example of the subjects.An 80-year-old male patient had been under observation for dry-type AMD in his left eye. There was history of severe vision loss in the right eye due to a disciform scar secondary to AMD since 1995. VA in his left eye had been 20/25 with confluent soft drusen in the macula. In September 2006, he noticed sudden visual loss to 20/200. Ophthalmoscopy disclosed subretinal hemorrhage in the macular region . FluoresIn this small case series, intravitreal injection of expansile gas with or without rtPA injection and followed by bevacizumab, proved successful in anatomical displacement of SMH and was associated with visual improvement. Mean preoperative VA was 1.28±0.27 logMAR which improved significantly to 0.57±0.33 logMAR at 12 months.The visual outcome of AMD patients with submacular hemorrhage is typically poor, particularly in eyes with thick blood clots or those involving large areas of the macula and in the presence of a choroidal neovascular membrane. The size of the CNV and the amount of remaining SMH affect the volume of the disciform scar. Four reports have been published describing the natural course of submacular hemorrhage.The Submacular Surgery Trial investigated AMD patients with SMH and reported decrease of two or more lines in VA in 64% of eyes with untreated SMH after 36 months of follow-up, as compared to 51% of eyes undergoing mechanical removal of the underlying CNV and SMH by pars plana vitrectomy.HeriotPreviously, additional intervention with photodynamic therapy (PDT) was employed if the SMH was shifted successfully from the fovea and the underlying CNV became visible. Bakri et alAntiangiogenic agents have opened a new approach toward effective and early treatment of choroidal neovascularization. The consecutive application of antiangiogenic agents treats the CNV which is the underlying cause of SMH. Stifter et alMeyer et alIn the present series, intravitreal expansile gas injection, with or without intravitreal rtPA injection, was successful in achieving anatomic displacement of submacular blood. There has been some doubt regarding the role of rtPA in the management of SMH, in particular, whether intravitreal rtPA can access the subretinal space in sufficient quantities to induce clot liquefaction. There are several small clinical series to suggest that a gas bubble alone is enough to displace submacular blood and the role of rtPA remains uncertain. It has been shown that albumin, a protein comparable in molecular size to rtPA, can diffuse across the intact retina. Furthermore, tenecteplase, a synthetic variant of rtPA with almost identical molecular weight and size, has been shown to be able to penetrate through all layers of the retina following intravitreal injection in a porcine model.Expansile gas and rtPA physically shift the SMH away from the fovea and minimize scar formation. Anti-VEGF treatment can be given prior to, or simultaneous with pneumatic displacement of the submacular hemorrhage. Meyer et alIn summary intravitreal expansile gas injection, with or without recombinant tissue plasminogen activator, followed by intravitreal bevacizumab injection seems to result in visual improvement in eyes with SMH secondary to neovascular AMD which may otherwise have a poor visual outcome.


The influences of polysaccharide concentration, sodium chloride concentration, autoclaving time, refrigerated time, and pH value on RS3 formation were discussed. Except for sodium chloride’s persistent restraint on RS3, the others all forced RS3 yields higher at first, but lowered it after the peak value. The influencing sequence of these impact factors was: sodium chloride concentration > polysaccharide concentration > autoclaving time > refrigerated time > pH value. The results also proved that in the three gums, KGM plays the most significant role in RS3 changing. It was concluded that the incorporation of each of these three gums into starch, especially KGM, results in an increase or decrease of RS3 under different conditions. This phenomenon could be taken into consideration when developing starchy food with appropriate amount of RS3.Retrograded starch (RS Resistant starch (RS) is the sum of starch and the products of starch degradation not absorbed in the small intestine of healthy individuals . RS is r1–4 [3, retrograded starch, is of particular interest, because of its thermal stability [The RS is classified into four different types, named RS1–4 . RS3, retability . This altability .3 is derived from natural starch during processing. On the one hand, it is a potential food additive, considering the variety of physiological function it possesses. On the other hand, starch retrogradation plays an important role in the processing technology, quality, taste and shelf life of starchy food, in most of which RS3 is unwanted. Thus, it is essential to explore both the impact factors and ways to restrain or boost retrogradation.RSD-(1→4) linked glucoses; and amylopectin, the branched glucan, composed of approximately 4000 glucose units with branches occurring as α-D-(1→6) linkages [Starch is comprised of two molecular types: amylose, the straight chain polyglucan, composed of approximately 1000, α-linkages .3, the starch granule is completely hydrated. Amylose leaches from the granules into solution as a random coil polymer. Upon cooling, the polymer chains begin to re-associate as double helices, stabilized by hydrogen bonds [In the formation of RSen bonds ,6. The ien bonds . T < 100 °C) gelation and crystallization within the amylose matrix. Long-term increases in the modulus of starch gels are linked to a reversible crystallization, involving amylopectin within the granules on storage [Starch gels are considered as composites containing gelatinized granules embedded in an amylose matrix. The starch retrogradation can be divided into two steps, the short-term and the long-term retrogradation. The short-term development of gel structure and crystallinity in starch gels is found to be dominated by irreversible  propagation, i.e., growth of crystals from the nuclei formed; and (3) maturation, i.e., crystal perfection or continuing slow growth. The extent to which these processes occur is clearly dependent on the temperature of the crystals (Tm); it increases with increasing extent of undercooling (Tm–T) or decreasing temperature. At temperatures below the glass transition temperature (Tg), the nucleation rate is negligible; the system is “frozen”. The propagation rate is 0 at T < Tg because diffusion does not occur at such temperatures. At high temperatures, diffusion increases and so does the rate of propagation. At temperatures above Tm, the propagation rate is also 0. The maturation rate is dependent on temperature in a way similar to that of the propagation rate [In general, crystallization consists of three steps ,12,13,14ion rate . The oveTg later might facilitate the RS3 formation. The promotion of temperature cycle on RS3 formation has been studied by some researchers [It could be concluded from the theory of crystallization discussed above that placing starch paste at low temperature first and raising the temperature over earchers . The rate and extent of retrogradation is dependent on both starch botanical sources and treating conditions . It is kIn the food industry, there are several kinds of hydrocolloids used to change the structural organization and rheological properties of starchy food . The hydMost works on starch retrogradation are conducted on starch molecule structure, amylose/amylopetine ratio, incubation time and temperature. Limited reports on starch-gum retrogradation behavior have been published ,21,22,23Amorphophallus konjac [Zhongyou 903  from the first crop of 2005 was obtained from Jingzhou District Agriculture Improvement Station, Hubei, China. Rice samples were dehulled, polished and ground in order to obtain rice flour. The rice flour was treated in the same method of a former research  to obtais konjac . 2HPO4(AR), NaH2PO4(AR) from Sinopharm Chemical Reagent Co. . They were all used without further purification.Highacyl gellan (H) KelcogelLT100 was provided by CP Kelco Co. . K-carrageenan (BR) and calcium chloride (AR) were purchased from Wuhan Tianyuan biomaterial Co. . Alpha-Amylase, pepsase and dried glucamylase were purchased from Sigmae-Aldrich Chemical Co. . HCl , NaOH, KOH , NaCl , NaStarch solution was prepared by mixing starch with deionized water at a concentration of 2% (w/w) by mechanical stirring. The solution was pre-gelatinized at 100 °C for 10 min by hot bathing. Then, a different amount of gum as well as NaCl was added to the solution, and the pH value was adjusted by 0.1 M HCl. Afterwards, the mixture was autoclaved  for a different time, and cooled to room temperature before being stored at 4 °C for a number of hours. After that, the gel was left at room temperature for 6 h, dried at 80 °C for 18 h, smashed, and sifted through a 63 μm screen.et al. [The resistant starch content was measured using the procedure of Goñi et al. . The metet al. .3 was formed.Starch was mixed with water, via high temperature treatment: the starch granule was completely hydrated. Amylose leached from the granules into solution. Following processing, the polymer chains began to re-associate, stabilized by hydrogen bonds, and RS3 content was counted and recorded in The concentrations of the three polysaccharides were set at five different levels , respectively. The mixed samples were adjusted to pH 7, added 10% NaCl, autoclaved for 60 min, and refrigerated for 12 h. Every experiment was repeated three times. After the reaction, RS3 content was counted and recorded in The concentrations of the three polysaccharides were set at 0.2%. The mixed samples were adjusted to pH 7, and 0%, 5%, 10%, 15% and 20% of NaCl were added respectively, autoclaved for 60 min, and refrigerated for 12 h. After the reaction, RS3 content was counted and recorded in The concentrations of the three polysaccharides were set at 0.2%. The mixed samples were adjusted to pH 5, 6, 7, 8, 9 respectively; 10% of NaCl was added, autoclaved for 60 min, and refrigerated for 12 h. After the reaction, RS3 content was counted and recorded in The concentrations of the three polysaccharides were set at 0.2%. The mixed samples were adjusted to pH 7, 10% of NaCl was added, autoclaved for 20, 40, 60, 90 min respectively, and refrigerated for 12 h. After the reaction, RS3 content was counted and recorded in The concentrations of the three polysaccharides were set at 0.2%. The mixed samples were adjusted to pH 7, and 10% of NaCl was added, autoclaved for 60 min, and refrigerated for 6, 8, 12 h, respectively. After the reaction, RS3 formation was dependent on polysaccharide concentration. The RS3 content kept increasing with the gum concentration rising from 0% to 0.2%, but at higher concentrations, a decrease was observed. This trend indicated that with the increasing concentration of polysaccharides from 0% to 0.2%, the reaction between starch and gum facilitated RS3 formation, however, when the concentration was over 0.2%, the solution viscosity was too high for RS formation. The RS3 formation were different. For concentrations of polysaccharides lower than 0.1%, the RS3 content did not change remarkably among the three polysaccharides, but all increased slightly. RS3 content with the presence of KGM increased more significantly compared with the other two samples, while the polysaccharide concentrations were between 0.1% and 0.2%. The addition of CA had the smallest effect on RS3 formation. Furthermore, there was a sharp decrease of RS3 content with the presence of KGM within polysaccharide concentration from 0.2% to 0.3%, while a slow decrease with the presence of the other two gums. When the concentration of polysaccharides was more than 0.3%, no remarkable change of RS3 content was observed.It could be observed from etc.) being kept equal, the different characteristics of the gums co-existing with the starch could be employed to explain the results. According to the former research of the same team [3 content was bound up with the properties of the starch-gum gel. During storage, the lower the viscosity of the gel, the more violent was the molecular motion. Violent molecular motion was in favor of the formation of a more compact double helix structure. Contrariwise, high viscosity blocked the molecular motion of the gel, and resulted in less crystal nucleus. As has been found with increasing KGM concentration, the adhesiveness of the mixed gel did not change significantly [The interaction between polymers is a very complex process, which could be influenced by a range of factors. With other conditions : sodium chloride concentration, polysaccharide concentration, autoclaving time, refrigeration time, and pH value. In addition, different gums display various effects on RS3 formation. In general, the KGM-starch mixture produced the maximum RS3, comparing with the other two gums under the same experimental conditions. The RS3 produced with each of the three gums is reversible, indicating that the properties might be different from that produced without the gums.The present study shows that the five factors which have been investigated above can influence the RSFrom the synthesization of this research and the other researchers ,21,22,233 formation. With an increasing demand for starchy foods with different qualities, it is likely that these functional ingredients will become more important to the starch food industry. However, interactions between starch and gums during autoclaving are not well understood. The way in which these gums affect the starch retrogradation requires further investigation.The present data demonstrate that it is possible to add gums to starchy food formulations to increase or decrease RS


The CorneaL GrAft Thickness Evaluation (COLGATE) system was recently developed to facilitate the evaluation of corneal graft thickness from OCT images. Graft thickness measurement can be a surrogate indicator for detecting graft failure or success. The purpose of this study was to determine the reproducibility of the COLGATE system in measuring DSAEK graft area between two observers.This was a prospective case series in which 50 anterior segment OCT images of patients who had undergone DSAEK in either eye were analysed. Two observers  independently obtained the image analysis for the graft area using both semi automated and automated method. One week later, each observer repeated the analysis for the same set of images. Bland-Altman analysis was performed to analyze inter and intra observer agreement.There was strong intraobserver correlation between the 2 semi automated readings obtained by both observers. (r = 0.936 and r = 0.962). Intraobserver ICC for observer 1 was 0.936 (95% CI 0.890 to 0.963) and 0.967 (95% CI 0.942 to 0.981) for observer 2. Likewise, there was also strong interobserver correlation (r = 0.913 and r = 0.969). The interobserver ICC for the first measurements was 0.911 (95% CI 0.849 to 0.949) and 0.968 (95% CI 0.945 to 0.982) for the second. There was statistical difference between the automatic and the semi automated readings for both observers . The automatic readings gave consistently higher values than the semi automated readings especially in thin grafts.The analysis from the COLGATE programme can be reproducible between different observers. Care must be taken when interpreting the automated analysis as they tend to over estimate measurements. Descemet stripping automated endothelial keratoplasty (DSAEK) is rapidly becoming an alternative to penetrating keratoplasty (PK) for patients with corneal endothelial failure . The proOptical Coherence Tomography (OCT) is an infra red light imaging device that captures micrometer resolution, three dimensional images of the anterior segment . ExampleUsers are able to quantify the images captured with the OCT using the built-in caliper software. For corneal graft thickness measurements the user can manually adjust the calipers to obtain thickness measurements at different locations along the graft. With individual point caliper measurements, the user only obtains information at a selective point on the graft and information about the complete graft thickness profile is not obtained. In order to circumvent this problem, a semi-automated system, the COrneaL GrAft Thickness Evaluation (COLGATE) software program was developed to facilitate the evaluation of corneal graft on OCT images. The COLGATE system  automatiThe aim of this study was to evaluate the inter-observer and intra-observer reproducibility of DSAEK graft area measurements using both automated and semi-automated methods of the COLGATE program. The results from this study allows for validation of the software for future studies.In this retrospective study, 50 high resolution cornea OCT images of 23 patients who had undergone DSAEK in either eye were obtained from a common data base. The data base encompasses all anterior segment OCT images which are kept in the hard disk drive of the OCT machine These 50 images were selected as they had the best quality images with both cross-sectional ends of the DSEK graft seen. There were 10 females and 13 males ranging in age from 42 to 83 years old, with mean age of 62.7 years old +/− 17.6. Of these, 52.2% were Chinese, 17.4% were Malays, 17.4% were Vietnamese, 8.7% were Indians and 4.3% were Eurasians. The study had the approval of the hospital’s Ethics Committee  and was conducted according to the tenets of the Declaration of Helsinki.The AS- OCT   images of the anterior chamber were obtained using high resolution cornea images in a completely dark room with no windows and the only lighted areas were the fixation target which equates to 20 lux illumination. To obtain the best quality image, the examiner adjusted the saturation and noise and optimized the polarization for each scan during the examination so as to obtain good discernible images with high signal to noise ratio. The patients were instructed to fixate at the external fixation light to ensure that they were looking straight ahead. The patients’ eye lids were kept open so that they did not block the 10-mm diameter corneal mapping. The operator adjusted the software system to position the vertex at the center of the AS-OCT image and to maximize the vertex reflection. The images were obtained by the same operator for all 50 eyes. All scans were taken between 10 AM and 4 PM to minimize the effect of diurnal variation on cornea thickness. More than one horizontal scan was performed but the best quality scan was selected by the technician performing the scan, for measurements.The images were processed through the COLGATE program that analyses the images via four main steps as previously described . The firThe definition of reproducibility used in this study was based on definitions by the International Organization for Standardization ,9. Two oBland-Altman analysis was performed to analyze inter and intra observer as well as between automated and semi-automated agreement using MedCal Version 12.3.0 . The reproducibility of the above parameters was calculated in terms of limits of agreement . Paired t-tests were used for the differences between observer measurements.Both observers were able to define the outline of the graft for all 50 images and calculate the graft area in an automated and semi automated method, on two separate occasions.The first and second sets of readings were similar within each observer as evident by strong linear correlation coefficient values (r = 0.936 and r = 0.962) as well as intraobserver correlation coefficient values (ICC). The intraobserver inter class correlation coefficient (ICC) for observer 1 was 0.936 (95% CI 0.890 to 0.963) and observer 2 was 0.967 (95% CI 0.942 to 0.981). This implies that overall the two sets of readings made by each observer not only strongly resemble each other  but each observer was able to obtain a point to point similarity between the two sets for readings of the calculated graft area for all 50 images (ICC). The mean difference of each of the calculated graft areas by the semi automated method, was expressed as the mean of the limits of agreement (LoA mean) and this was smaller for observer 1 ie −223.8 um2 (−4340.6 to 3893.1) than for observer 2 at −3477.5 um2 (−6695.5 to −259.5).  was reflected in the linear correlation values (r = 0.913 for the first set and r = 0.969 for the second set) and there was no statistical significance in the calculations made between the two observers for each set of readings. This was further supported by the interobserver interclass correlation coefficient value (ICC) for the first set of measurements between observer 1 and observer 2 (0.911 (95% CI 0.849 to 0.949)) and the second set of measurements 0.968 (95% CI 0.945 to 0.982). This means that there was point to point agreement of the calculated graft area obtained between observer 1 and observer 2 for all 50 graft area calculations for both sets of readings. The difference of each calculated graft area  by observer 1 and observer 2 from the mean of both their calculations is again expressed as the mean LoA. The mean LoA for the first set of readings (between M1 and B1) was 3364.4um2 and even was even smaller for the second reading (between M2 and B2) at 110.6 um2  were compared. In the first set (automated versus M1), there was statistical difference in the mean graft area calculated by the automated method (195474.6um2) and semi-automated 151619.4um2 by observer 1(p = 0.006). The area calculated by the automated method and the first set of calculations made by the first observer (M1) were not correlated r = −0.083. The LoA mean in this first group was 43855.14 um2 implying that 95% of all calculated graft areas of the 50 images using the automated and semi automated method were found to be far from the mean of the calculated automated and semi automated graft area.In the second set, the graft area calculated by the automated method was 195474.6um2 and semi-automated 151843.2um2 (M2) (p = 0.006). Again, the two sets of results were not correlated (r = −0.08) and the large LoA mean of 43631.38 um2.Observer 2 also had 2 sets of graft area calculations compared with the automated method. For the first set of readings done by semi automated method (B1), observer 2 obtained 148255.0 um2 for the mean graft area while the automated method had a reading of 195474.6um2 (p = 0.003). The calculated area of each image obtained by the automated method and observer 2 were not correlated (r = −0.08). Likewise, the large LoA mean of 47219.5 um2 implies that 95% of all calculated graft areas by automated and semi automated method (B1) were far from the mean of the automated and semi automated graft area values (B1). For the second set of calculations made by observer 2 (B2), observer 2 obtained 151732.6um2 and this was statistically significantly different (p = 0.006) from the automated value. Once again, there was a negative linear correlation of r = −0.05 and a correspondingly large limits of agreement between automated and B2 values (LoA mean 43742 um2). Table There is much subjective variation in the software caliper placement by users of the ASOCT, and inter-observer variations of measurements have been shown to have a SD of 18.0 - 20.2um at +1.0 mm and −1.0 mm from the centre of the cornea.* Recently we compared the interobserver and intraobserver variation of LASIK flaps measured using a time domain and spectral domain machine . The int2 measured by the semi automated method had more than double the measurement by the manual method. This may be due to the fact that the boundary between the anterior graft surface and the underside of the recipient stroma might not be as easily discernible in thin grafts by the COLGATE program. The authors believe that using the semi automated method to calculate the graft area would be better in all grafts.In this study, we found that the COLGATE program was a highly reproducible tool for graft area measurements and consistent graft area calculations could be obtained both inter and intra-observer from every scan. This was evident by the strong linear correlation coefficient, intraclass and interclass coefficient and the small range of limits of agreement. It was observed that the automated method gave consistently higher values than the semi automated method and this was especially pronounced in thin grafts. In particular graft areas of less than 80000um# used in glaucoma for evaluating the optic nerve head or the Artemis high frequency ultrasound system [We found a higher linear correlation of the graft area measurement made between observers  using the COLGATE program than the previously reported inter-observer correlation on the same time domain ASOCT  [d system .The COLGATE system may be used as an objective method for ophthalmologists and researchers to obtain graft area measurements. This raises the possibility that DSAEK surgeons may then have a program to preoperatively predict the maximum donor diameter of the graft to be inserted so as to enable transfer of the maximal amount of donor endothelial cells, and with a chart including a range of individual donor thicknesses, be able to select and decide on the exact diameter required at the time of surgery taking into account the actual thickness of the donor tissue supplied for the case, without the fear of encroaching into the chamber angle and risking donor iris contact at the periphery.Currently, graft thickness is measured using the software calipers on the Visante ASOCT (as well as other OCT systems) at a single point. However, as most grafts are irregularly cut, the central thickness does not provide a good estimate of the entire graft thickness . There iThere are other uses of this software. Its use can be extended to evaluating penetrating keratoplasty grafts, as well as anterior and deep anterior lamellar grafts. Future software enhancements will also allow it to be possible to measure the anterior and posterior curvature of the graft, which are useful parameters in assessing post graft refractive evaluation . Current2, volume = area x size of trephine (ie 8.5 mm) = 170,000um3 It is also not sufficient to simply rely on the automated readings as we found that the semi automated readings gave a better and consistent graft area calculation.There are some limitations to this program. Though the graft area is a better surrogate for graft thickness than single point measurements as it incorporates the entire graft, the graft volume would be a more precise parameter than area. However, this would require a three-dimensional imaging acquisition of the corneal graft, which currently is not available. An estimation of graft volume may be calculated from our values by multiplying the graft area by the graft diameter which will be known to the surgeon implanting the graft e.g. if area is 20000 umLastly, it would also be interesting to demonstrate how image quality changes the inter-observer and intra-observer reproducibility of the measurements.This paper shows that the intra-observer and inter-observer repeatability from the COLGATE program is highly reproducible, and could be used clinically to determine the ideal diameter of a donor lenticule for DSAEK surgery. However care must be taken when interpreting the automated analysis as it tends to over estimate corneal graft area, especially in thinner grafts. Nevertheless, the semi-automated program is useful in evaluating the graft area which in turn is a better indication of the graft thickness and hence graft function. Further studies evaluating graft thickness and graft refractive power longitudinally postoperatively using this software are planned. Knowing the expected hyperopia from the calculated graft thickness can help the surgeon determine the refractive end point especially when performing a combined DSEK with phacoemulsification.aVisante Operation Manual. Jena, Germany, Carl Zeiss Meditec AG, 2006;appendix D-6:158.bhttp://www.heidelbergengineering.com.The authors report no conflicts of interest.JM, DT: Initiating the study and tutoring MW and AC. MW and AC contributed to the acquisition of images using the software. MW: Writing of the manuscript. H Htoon assisted with statistical analysis. JLee, J Cheng and B Liu: contributed to the analysis and creation of the COLGATE software. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/12/25/prepub


Descemet membrane endothelial keratoplasty (DMEK) is a corneal surgical technique which selectively replaces the damaged posterior part of the cornea with a healthy donor graft retaining the rest of the tissue intact. There is a need to validate and standardize the donor tissue before grafting due to certain issues that can lead to consequences such as graft failure due to poor endothelial cell count, higher mortality, detachment of the graft, or increased surgical expenses, time, and effort. Thus, prospective potential surgeons and eye banks should now aim at developing new improved surgical techniques in order to prepare the best suited, validated, precut, preloaded, and easy to transplant tissue to reduce pre- and postsurgical complications. This could be achieved by defining parameters like graft thickness, accepted mortality threshold of the endothelial cells, and behavior of grafts during preservation and transportation along with using more sophisticated instruments like microkeratome and femtosecond lasers for graft preparation. Thus, a rapport between the eye banks and the surgeons along with the advanced instruments can overcome this challenge to find the best possible solution for endothelial keratoplasty (EK). The cornea is the anterior part of the eye globe. It is an avascular tissue which is directly exposed to the external environment. It focuses the image by refracting the light to the retina through a lens. Hence, it should be clear and well maintained for optimal visibility . The corCorneal lamellar keratoplasty (LK) is a surgical technique that allows preserving healthy portions of the cornea while selectively replacing the dysfunctional segments. The best action to treat corneal disorder is a replacement of the damaged recipient cornea  with a healthy donor corneal tissue. Deep anterior lamellar keratoplasty (DALK) is a surgical technique that is considered the best for treating the patients with anterior layer  disorders. Descemet's membrane endothelial keratoplasty (DMEK) is currently pursued to treat the patients with endothelial dysfunction. With time, Descemet's stripping automated endothelial keratoplasty (DSAEK) has evolved drastically with an instrument such as a microkeratome, which is more standardized, efficient, and safe to create corneal grafts or lenticules. The different techniques for specific layers are illustrated in ALK targets the replacement of the damaged anterior segment (epithelium and part of stroma) of the recipient's cornea with the anterior part of the healthy donor tissue. The deeper layers (posterior) of the recipient cornea, specifically the endothelium and the Descemet's membrane, are left intact which reduces the risk of rejection and therefore has a distinct advantage over PK. Deep anterior lamellar keratoplasty (DALK) replaces both, the epithelium and most of the stroma with the donor tissue. This is favorable for those disorders which affect the anterior segment of the cornea –9. AccorEK selectively replaces the diseased corneal endothelium with healthy donor tissue through a small limbal incision while retaining the healthy anterior part of the patient's cornea. This surgical technique has multiple advantages over PK as the recipient cornea remains structurally intact and resistant to injury. In addition, since it is a suture-less surgery, the results lead to quick rehabilitation and better visual outcomes. In general, the recipient eye is maintained much stronger as compared to PK. The different types of EK currently pursued are the following. This procedure is the first hand/primary technique with excision of the posterior recipient stroma and endothelium with small curved scissors and trephine. The donor tissue is folded for insertion through a small incision near the limbus region.  The posterior membrane which includes the Descemet's membrane and the endothelial cells is excised and transplanted. The thinnest possible lamellar graft is transplanted with the intention of better and faster visual recovery and outcomes. Majority of the tissues are prepared today with an air bubble technique. The cellular mortality after the preparation of the graft can be a major concern if the DMEK is created by stripping it off manually. A recent study described by Dapena et al. showed a no-touch technique for DMEK surgery. As per this study, the technique could provide best corrected visual acuity (BCVA) of 20/25 or more with a good endothelial cell density after 6 months from surgery. The steps include incision and descemetorhexis (excision of Descemet's membrane from the recipient cornea), preparation and implantation of DMEK graft followed by orienting, unfolding, centering, positioning, and fixing the DMEK graft. This technique claims to be more standardized with near complete visual recovery and minimal endothelial cell loss. It further explains that approximately 95%of the cases may gain a BCVA of 20/40 or better and 75% may attain 20/25 within 6 months post-op . Another report cited a combination of two procedures, that is, preparation of “no-touch” DALK and DMEK grafts from the same donor. The rolled tissue was placed on a soft contact lens which was used for trephination of the endothelial graft using a custom-made trephination system. This technique claims to produce undamaged grafts with better handling of the tissues especially for thin Descemet's membrane. As the undamaged anterior cornea could also be used after separating the Descemet's membrane, this method increases the availability of the donor tissue by using two different grafts from the same donor. No clinical signs of graft dysfunction, primary/secondary graft failures, or graft detachments were observed. The endothelial cell density and the relative mortality showed no significance in terms of cell loss before and after technique .Descemet endothelial graft (DEG) can be isolated as in the studies described above or isolated after pneumatic dissection  using air bubble technique and preserving the lenticules for 7 days in organ culture. In the latter studies, the anterior stroma was removed using the microkeratome followed by air injection to separate the Descemet's membrane and the stroma and then attached to a silicone weight using a scleral ring. This technique showed that the DMEK tissues can be pre-prepared in the eye banks and can be preserved with a minimum endothelial cell loss . n = 5; average stromal residue = 12.45 micrometers). This concludes that although with a possibility of creating a thin lenticule, the presence of stroma indicates that the technique should not be termed as DMEK but a very thin DSEK [However, a similar study showed that although using air as a medium to create the bubble could be useful, the lenticule demonstrated the presence of residual stroma in all the tissues that were harvested  was marked with letter “S” and the rest of the stroma was amputated. This technique showed less endothelial cell loss and claimed to be a potential method as it required no special surgical instruments. Although the endothelial cell density was noted, the mortality after the lenticule preparation was not recorded. The ECD 1 year post-op showed approximately 44% loss. Therefore, the hypothesis could be (a) the cell loss was due to the recipient acclimatization post-op or (b) the transplanted cells were damaged or dead after graft preparation. The mortality checks therefore become an important parameter and an issue that needs to be highlighted for the DMEK surgeries. Hence, determining the mortality after graft preparation is recommended . In conclusion, there are several methods that have been introduced which have different approaches to retrieve the DEG, preserve and supply as either precut tissue from the eye banks or preprepare at the surgical theatre. However, there is a lack of a standardized method which can repeatedly prove the reduction of risk or complications that are usually seen due to unidentified parameters like mortality and other risks or complications such as graft failure due to detachment or poor endothelial cell count post-op. Even with its limitations, EK has succeeded PK as the first choice of treatment for endothelial dysfunction due to its advantages like quick and efficient surgery with reduced manipulation, low surgical risk, and better visual outcome. Innovations in EK with modification in donor preparations have broadened its use and improved intraoperative ease, and reduced postoperative complications have therefore been responsible for its emerging popularity  as shownIt is less traumatic as compared to manual procedures that include scissors and trephines. A mechanical microkeratome is used to simplify the donor tissue dissection, thus, making the procedure more standardized and easy with lesser damages to the prepared graft. Furthermore, as the anterior corneal surface is not manipulated, it does not result in any of those refractive errors that is usually seen after PK. However, it might show a slight hyperopic shift due to changes in the curvature or astigmatism. More recently, FS lasers have also been used for donor tissue dissection . This meWhere Laser-Assisted In Situ Keratomileusis (LASIK) helps to correct the refractive errors, improved FS laser engines are proving to be useful for lamellar and cataract surgeries . EarlierOther advantages of the FS laser include (a) precise cuts at specific sites; (b) higher reproducibility; (c) reduced dissection issues; (d) standardized procedures with specific thickness; (e) establishing safe and reliable procedure due to satisfactory outcomes with smoother stromal surfaces which is important for long-term visual outcomes . Both saμm become more rough and irregular due to reduced laser beam focus. It is believed that setting the spots closer together with more focal energy and adjusting the spot and line separation can help to create a smoother dissection even while creating deeper cuts [Rousseau et al. showed that the issues arise with the donor corneas when determining the optimal amount of energy for a lamellar cut. The optimal setting should be enough to penetrate deep into the posterior stroma, overcome diffraction, and prevent any keratocyte activation or inflammatory reactions . Posteriper cuts .iFS laser seems advantageous when transplants involve thin button of anterior corneal tissues. The interface gets rough when the laser reaches deep towards the corneal stroma for full lamellar cut . It has The 60 kHz FS laser allows closer spot and line separation with lower energy levels and results in smooth stromal interface also in deeper cuts which states that higher frequency with lower energy and closer spot and line separation can create smoother stromal bed surfaces , 20. BetDSAEK is a standardized method to perform EK, unlike DMEK. As it reduces the risk of complications and allows a better and faster recovery and visual outcomes, it has become a goldstandard amongst the eye bankers and surgeons. Although good results have been achieved against PK, many surgeons have speculated that DSAEK can perform even better in terms of visual acuity. However, the reason for poor performance is mostly based on a hypothesis related to the presence of a stromal interface. Therefore, the next challenge was to completely remove the stromal interface and create a DEG. In 2006, Melles introduced DMEK procedure where only the donor's DEG was stripped off, thus, removing most of the stromal interface. This procedure reported a number of patients with 20/20 or better, but did not exceed 40%. It was therefore concluded primarily that the stromal interference during the EK could not be the only reason of poor visual outcome post-op. Moreover, DMEK requires high surgical skills, tissue preparation followed by surgical time, unlike DSAEK. In addition, a high rate of tissue loss and detachment rate with a huge amount of graft failures have discouraged most surgeons to adopt this technique [Recent studies have demonstrated that DSAEK grafts that are thinner than 131 micrometers have shown 20/20 vision post-op. Moreover, hyperopization is reduced due to peripheral edges , 38. DSAμm of thickness. The endothelial cell density before and after preparation showed an average loss of approximately 3-4%, although the difference was not found significant. If it is proved that the residual stroma does not interfere with the visual outcome post-op, UT DSAEK could be the future due to its benefits that include standardization, lesser manipulation, or manual error [The microkeratome-assisted UT DSAEK preparation showed that double cuts can create lenticules with <100 al error .With the current studies, DMEK, which makes use only of the Descemet's membrane and the endothelium for transplantation, is not a standardized procedure and due to the requirement of high surgical skills only a small number of surgeons are capable of performing it. Moreover, the major drawbacks include the preparation, manipulation of donor material, unpredictable complications, and graft failure rates. Although it has a <40% success rate, up to 16% of graft failure before surgery, approximately 63% of cases with detachments and 8% with primary graft failure , it is sAs we speculate, the eye banks will play an important role in the near future for the development of new surgical techniques in collaboration with the clinicians. One of the issues for eye bankers today is that there is no standard or threshold limit for the requirement of the viable endothelial cell count for critical surgeries like DMEK. This creates a lot of confusion, when a surgeon demands a precut tissue for DMEK or when a surgeon is preparing the graft before surgery. Moreover, as mentioned earlier, the majority of surgeons do not calculate the mortality or the endothelial cell density after the tissue preparation, which addresses a challenge to the eye bankers to standardize the mortality issue. This also results in a false positive post-op endothelial cell survival study although the results for surgical success and visual recovery are documented to be positive. Thickness: can thinning the tissues to the minimum (DEG) be really useful in terms of visual outcome?Viable endothelial cell density: should the surgeons evaluate the mortality or the viable endothelial cell density after DMEK graft preparations before surgery?Standardization: can a standardized and validated pre-cut, preloaded tissue help to reduce the risk, time, and cost of the surgery? Thinning the tissues using UT DSAEK has provided better results, but needs to undergo a strong confirmation to practically prove the repeatability and long-term beneficial effects of this procedure. If thinning the tissues can create a more suitable visual outcome, then the amount of thickness and the detachment or other associated risks should be justified. Moreover, thinning the tissue can result in unwanted effect of rolling the DEG on itself which can decrease the cell viability. A perfect DMEK/DEG usually has a thickness of 15–30 micrometers which is not enough to hold the lenticule without getting damaged. Therefore, it is very important to understand and validate the mortality threshold for the DEG. This will help to prepare a pre-cut DMEK/DEG in the eye banks and increase the quality control levels, save time, cost, and risks associated with the graft failure due to surgeon preparatory mistakes. Therefore, we believe that a more standardized, validated and ready to transplant tissue should be the future of DMEK surgery. Moreover, the recent advances in the use of FS lasers for donor graft preparation could be advantageous in terms of increasing the stromal interface smoothness and cuting it with more ease; however, it might be more expensive than the conventional techniques. Furthermore, it is also important to validate the procedure for DMEK graft preparation using FS lasers in terms of energy/frequency levels and thickness of the required graft. In the future, use of fibrin glue could prove beneficial for sampling, handling, and transporting the DMEK tissues with ease of surgery, although the pros and cons need to be identified if used in vivo [Thus, in general, the possible future challenges and the key issues which need to be identified and demonstrated for the standardization of the EK procedures could be the following. in vivo . According to a study, if the cost analysis of surgeon-cut and the eye-bank cut donor corneal tissue for EK is valued, then the cost per surgeon-cut donor corneal tissue decreases if the number of cases performed increases per year. Excluding other factors such as opportunity costs, the eye bank processing charge is almost equal to the expenses associated with a surgeon-cut cornea if the surgeon was to perform approximately 15 cases/year. The microfinance study was based on costs of equipments, consumable supplies, labor charges, building space, and risk of attempted damage . Even ifThus, we envision that standardizing the posterior lamellar graft preparation methods will reduce unnecessary manipulation of the tissue in the operating theatre and reduce the high surgical skill or risk quotient. In the near future, the DEG could be supplied as pre-cut tissues which would reduce the overall intervention costs and save time. A recent study has shown a moderate decrease in endothelial cell density if the DEG is left in storage under organ culture, which concludes that a pre-cut DMEK preservation is possible for future transportation . The fin