 cell membranes were subsequently stained with wheat germ agglutinin conjugate WGA-CF ® 594 (Biotium, Fremont, US) at a concentration of 5 µg/ml in Hank's Balanced Salt Solution (HBSS; Lonza) for 10 min at room temperature, followed by washing twice in PBS. Nuclei were stained with DAPI (200 ng/ml) (Sigma) by 15 min incubation at room temperature. After two washes with PBS, the slides were mounted with 50% glycerol in PBS before fluorescence microscopy using a Leica DMI6000 (Leica, Wetzlar, Germany). The internalisation level of tested strains was calculated as a ratio of invaded C. jejuni and infected host cells, which were automatically counted by the ImageJ software as previously described [27]. Briefly, we used ImageJ to outline interested signals by thresholding and then identified particles with a specific size range. Afterwards, the particle counting was automatically measured by the Region of Interest (RoI) manager.

Cell viability determination
To study effects of C. jejuni on HT-29/B6 viability, Calcein-acetoxymethyl/Hoechst staining was performed. Calcein-acetoxymethyl (Calcein-AM; Biotium) has already been described as a green fluorescent indicator of viable cells in cytotoxicity assays [29,30]. Hoechst 33342 (Thermo Fisher Scientific) (hereinafter referred to as Hoechst) is used as a blue fluorescent marker of the nuclei of all cells [53]. Viability of HT-29/B6 cells was investigated in 96 well plates (Sarstedt). After 7 days differentiation, HT-29/B6 with ~ 80-90% confluence were infected with wild type, ::Cj1492c and ::Cj1507c for 5 h and 24 h as described before. Infected monolayers were washed 3 times with PBS and incubated with 0.4 µM Calcein-AM at 37 ℃ with 5% CO 2 for 30 min. 5 µg/ml Hoechst was added to each well and incubated 15 min at room temperature followed by two washes with PBS. Fluorescence micrographs were subsequently captured with a Leica DMI6000 (Leica). The relative viability of host cells was calculated as percentage of the number of viable cells compared to the total cell numbers automatically determined using the ImageJ software as described above [27]. Presented results are calculated from three individual assays.

RNA isolation and RT-qPCR
For mRNA expression analysis, in vitro infection was performed as described above and HT-29/B6 samples were taken at 1, 5 and 24 h p.i. considering respective negative controls (non-infected cells). For this, cell monolayers were washed three times with PBS and lysed directly in each well of 8-well plates by addition of cell lysis buffer (Roboklon, Berlin, Germany). Total RNA was isolated with Universal RNA/miRNA Purification Kit (Roboklon) according to the manufacturer's protocol. The RNA quality and quantity was controlled using the Agilent 2100 Bioanalyzer with the RNA Nano Chips (Agilent, Waldbronn, Germany) and the DS-11 Spectrophotometer/ Fluorometer (DeNovix Inc., Wilmington, USA), respectively. Relative expression of mRNA coding for IL-6 and IL-8 was quantified by RT-qPCR as described earlier [54]. For normalisation of gene expression, ACTB and B2M transcripts were used as reference genes and relative gene expression was calculated as described earlier [54] by the ∆∆Ct method [55]. Experiments were performed in triplicate. Primers used in this study are listed in Table 1.Google Glass Is Limiting More then Liberating?
This is probably my first article posting on this forum.If I make some mistake then please don't mind guys and help me to correct it@@dykhatri.
​On the other hand other devices that comes with good camera with mic costing $300 as small part of Google Camera costing user around $1500.Google has to improve a lot since this product still having need for intelligence to deal with But Google will come up soon with solution this as limitation are constraints just for matter of time.
Nothing's perfect -- I don't blame Google...
Now that the idea its spread, samsung will grab it and make it better.
The Batteries issue is persist is all type of devices and whether it is Android or iOS or Windows.
We hope someone come up with batter option with improve batter performance.
"Here dream of yesterday become reality in today"Prelim year vs. Transitional --Stronger Applicant?
I have heard that either years are good prior to starting Ophthalmology PGY-2. But, what if you did not apply for match this year and plan on doing the prelim or transitional year prior to applying for the SF Match? Would you be a stronger applicant doing one or the other? Personally, I am more interested in doing the one year Prelim in Surgery: 1) This is because Surgery is my second choice and if I dont match into ophthal I can try to apply the PGY-1 year credits to a Categorical surgery program (i.e. get into a PGY-2 Cat Surgery program). 2) I have a feeling ophthal programs would see you as hard working and a better applicant than doing a trans year or a prelim medicine year.
Any advice on this? Am I on the right tract? Can it hurt me to do a prelim surgery year?
Where you do your prelim/transitional/internship year will not affect your competitiveness for ophthalmology. If you're interested in surgery as a second option, then I advise that you take this route.
I meant to say where and whether. The point is that the first year has very little bearing on your competitiveness.Afghanistan outclassed Hong Kong by eight wickets to reach the semi-finals of the Asian Games cricket tournament on Wednesday, skittling their opponents for 63 in a one-sided contest.
It sets them up with a clash against Pakistan for a place in the final.
Hong Kong, who won the toss and chose to bat in the Twenty20 contest, had no answer to Afghan opening pair Shahpur Zadran and Merwais Ashraf, slumping to 15-6 in the eighth over.
Zadran grabbed 2-9 and Ashraf took 3-8 in their allotted four-over spells as the Hong Kong batsmen looked out of their depth on a pitch offering some encouragement to the bowlers.
Hong Kong staged a mini-recovery, with Jamie Atkinson hitting a six over midwicket in his 16 off 28 balls but he was bowled by captain Mohammad Nabi when aiming to hit out, reducing the side to 41-7 in the 12th over.
They were bowled out for 63 in 17.4 overs, with Nadeem Ahmed the only other batsmen to reach double figures, top-scoring with 17.
Hong Kong's opening bowlers Aizaz Khan and Irfan Ahmed looked lively but failed to make the early breakthroughs required and Afghanistan reached their target in the 14th over for the loss of just two wickets.
Mohammad Shahzad and Nawroz Mangal both finished unbeaten on 20.
"We are a good bowling side. We have two fast bowlers and it's difficult to bat on this wicket as it's a seaming wicket," said skipper Nabi.
"It's not going to be easy to play Pakistan. They are a good team, especially on this wicket but we've played very well in the past three or four years and we have played together as a team."
Coach Rashid Latif, a former Pakistan Test player, said he was confident Afghanistan could beat Pakistan in Thursday's semi-final, predicting a low-scoring contest on the bowler-friendly pitch.
"On paper Afghanistan are stronger than Pakistan and I'm very hopeful because my players are mentally and physically tough.
"We have a good fast bowling and spinning attack and are agile in the field," he said.
Also through to the last four are Bangladesh, who will face either Sri Lanka or Nepal, playing later Wednesday.
Last year Afghanistan rose to the International Cricket Council's (ICC) Division One, narrowly missing out on a place in the World Cup to be held in India, Sri Lanka and Bangladesh next year.
They qualified for the World Twenty20 held in the West Indies earlier this year by winning qualifying rounds in Dubai.
Asiad: Afghanistan demolish Hong Kong to reach cricket semis
Afghanistan outclassed Hong Kong by eight wickets to reach the semi-finals of the Asian Games cricket tournament on Wednesday, skittling their opponents for 63 in a one-sided contest.Courtesy of Cynthia and that wonderful raffle, I had $50 to put toward an East Coast Ranchu order. I was out of town the first week and waited to order, but found some fantastic oranda babies to practice grooming, as well as the MOST ADORABLE RANCHU I’VE EVER SEEN and a majestic Thai oranda.
The babies! Eight Thai oranda babies, pre-color change. They’re so itty bitty. My kids have already decided to name one each and I’ll do the remainder, but not until I can tell them apart 🤣 We had a little rough start when they were mostly spitting food but I managed to get some bloodworms (I’M NOT HAVING MAJOR HIVES ANYMORE YAY!) into them and they’re tolerating my fluval Bug Bites now too, so long as the pieces are small. Still not bit fans